Biomolecules for development of biosensors and their applications

Sandeep K. Sharma a, Neeta Sehgal b, Ashok Kumar a,*

a Institute of genomics and Integrative Biology, Mall Road, Delhi 110007, Indiab Department of Zoology, Delhi University, Delhi 110007, India

Abstract

Biosensors are analytical devices incorporating biological materials such as enzymes, tissues, microorganisms, antibodies, cell

receptors or biologically derived materials or a biomimic component intimately associated with or integrated within a physico-

chemical transducer or transducing microsystem which may be either optical, electrochemical, thermometric, piezoelectric or

magnetic. The electronic signals produced are proportional to the concentration of specific analyte. A biomaterial may be any

material, natural or man-made, that comprises whole or part of a living structure or biomedical device, which performs natural

function. An essential component of molecular sensor is reagent layers. Creation of these layers require the immobilization of

recognition elements for the detection method. The recognition elements are biomolecules. Laboratory methods of immobilization

are numerous, but may not always appropriate for manufacture of biosensors. In the present article, we describe the use of various

biomaterials for biosensors as well as their availability.

� 2002 Elsevier Science B.V. All rights reserved.

PACS: 87; 87.14.)g; 87.14.Ee

Keywords: Biomaterials; Biomolecules; Biosensors; Enzymes; Immunosensors

1. Introduction

Biosensors are moving from the laboratory level to

field testing and commercialization in US, Europe and

Japan. Biosensors have potential for continuous andin situ application in fields such as medical diagnostics,

genetics and environmental monitoring. DNA micro-

chip technology is emerging on the horizon and being

designed to analyze gene expression patterns, genome

mapping and detect genetic mutations etc. Biosensors

such as nerve cells grown on a microprocessor, can

generate electrical signals in response to stimuli of toxins

present in the environment. Such type of technologiesare creating strategic, innovative links between elec-

tronic engineering and biological sciences [1].

Biosensors are analytical devices incorporating a bi-

ological material in an intimate contact with a suitable

transducer device that converts the biochemical signal

into quantifiable electric signals. A biologically derived

material or a biomimic intimately associated with or

integrated within a physicochemical transducer or

transducing microsystem, which may be electrochemi-

cal, thermometric, optical, piezoelectric or magnetic.

Biosensors usually generate a digital electronic signal,

which is proportional to the concentration of a specificanalyte or group of analytes (Fig. 1). Biosensors have

been used in a wide variety of analytical tools such as in

medicine, food, environment, process industries, secu-

rity, defence and diagnostics, etc. The emerging field of

biosensors seeks to exploit biology in conjugation with

electronics [2].

The biological component of biosensor can be

divided into two distinct groups i.e. catalytic and non-catalytic. The catalytic group includes enzymes, micro-

organisms and tissues, while the non-catalytic consist of

antibodies, receptors and nucleic acid etc. Various types

of transducers available for detection of analytes such

as electrochemical (amperometric, potentiometric and

conductometric), optical, colorimetric and acoustic etc.

The biological materials specially enzymes, multi-

enzyme complex, tissues, microorganisms, organelles,cell receptors, antibodies, nucleic acids or whole cells

(bacterial, fungal, animal or plant) are responsible for

recognition of the analyte [3]. Although, very minute

* Corresponding author. Fax: +91-11-27667471.

E-mail address: ashokcbt@rediffmail.com (A. Kumar).

1567-1739/02/$ - see front matter � 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S1567-1739(02)00219-5

Current Applied Physics 3 (2003) 307–316

www.elsevier.com/locate/cap

quantities of the biomaterials are required, but their

purity may play a vital role in reliability. The study ofnatural biopolymer is fundamental for understanding

how animal, plant cells and animal tissues function

and respond under different conditions. Knowledge of

three-dimensional structure of protein is necessary for

understanding and consequently controlling the modi-

fication of their biological activity, as might be required

for the proper design of a biosensor [4].

There is often a need for electrons to pass fromenzyme-based biological components to the amplifier or

microprocessor. Ferrocene represents the potential ways

of solving the problem. The cells in natural state provide

many examples of such transmission for example cyto-

chromes which are hemoprotein, whose main biological

function is electron or hydrogen transport by valency

change of their heme iron [5]. An essential component of

a molecular sensor is the reagent layer. Creation ofthese layers require the immobilization of the recogni-

tion elements for the detection method. In the case of

biosensors, this involve the biomolecules such as en-

zymes, antibodies, microorganisms, etc. (Fig. 2).

Various methods are available for immobilization of

biomolecules, but not always appropriate for manu-

facture of biosensors. The most commonly used bio-

material immobilization techniques for designing anddevelopment of specific sensors are physical adsorption,

entrapment, inter molecular cross-linking and covalent

binding:

1. Adsorption: Many substrates such as cellulose, collo-dion, silica gel, glass, hydroxyapatite and collagen are

well known to adsorb enzymes. Binding forces are

mainly due to hydrogen bonds, multiple salt linkages,

Van der Wall�s forces and formation of electron tran-

sition complexes.

2. Entrapment: If a polymeric gel is prepared in a

solution containing biomolecules, the biomolecules

become trapped within the gel matrix. The poly-acrylamide, starch, nylon and siliastic gel can be em-

ployed for the entrapment of the biomolecules.

3. Cross-linking: Intermolecular cross-linking of bio-

molecules by using bi-functional or multi-functional

reagents such as glutaraldehyde, hexamethylene

di-isocyanate, 1,5-difluro 2,4-dinitrobenzene and bis-

diazobenzidine-2,20-disulphonic acid, etc. These re-

agents can bind biomolecules to solid supports.4. Covalent binding: It is accomplished through func-

tional group in the enzyme which are not essential

for its catalytic activity. Usually, nucleophilic func-

tional groups present in amino acid side chains of

proteins such as amino, carboxylic, imidazole, thiol,

hydroxyl etc. are used for coupling.

The various methods used in immobilization ofmolecules are shown in Fig. 3 and their applications in

development of biosensors are shown in Table 1.

2. Biomolecules used in enzyme sensors

Enzymes are extensively used as biomaterials for the

development of biosensors e.g. glucose oxidase in glu-Fig. 2. Schematic representation of biosensor developed by attachment

of enzyme membrane to oxygen electrode.

Fig. 3. Immobilization methods of biomolecules.

Fig. 1. Schematic representation of biosensor.

308 S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316

cose sensor. Immobilized enzymes, together with elect-

rochemical sensors, are used in several instruments

available commercially. Kimble has designed a urea in-

strument using immobilized urease and ammonia elec-trode and a glucose measuring instrument using

insolubilized glucose oxidase and platinum (Pt) elec-

trode marketed in Europe in the trade name of Tech-

nicon. Yellow Springs Instrument Co. markets instrument

for measuring glucose with immobilized glucose oxidase

pad placed on a Pt electrode as well as the instrument

for lipase, triglycerides, cholesterol and amylase [6,7].

The biomaterials used in development of biosensors by

different industries are shown in Table 2. However,sometimes enzyme-based sensors are hampered due to

their stability at desired temperature. Besides, some en-

zymes even require cofactors for their optimum activity.

The problem of stability of the enzymes can be solved

by (i) using enzymes from extremophilic organisms or

Table 1

Immobilization procedures and their applications in development of biosensors [6–9,23,24]

Procedure Enzyme Applications

Adsorption on to graphite Glucose oxidase Glucose sensor

Adsorption on to calcium carbonate particle Nitrite oxidizing bacteria Environmental biosensor

Entrapment in a gelatin support LL-lysine oxidase Amino acid sensor

Entrapment between cellophane dialysis membrane and NH3 gas permeable

membrane

Asparaginase Asparaginase sensor

Glutaraldehyde mediated co-immobilization on Clark type electrode Alcohol oxidase and catalase Alcohol sensor

Glutaraldehyde mediated reaction with gelatin on to a CO2 gas sensitive

electrode

Lysine decarboxylase Amino acid sensor

Co-immobilization on nylon cloth of enzyme with electron acceptor

potassium ferricyanide

Glucose oxidase Glucose sensor

Enzyme on membrane over a platinum electrode Uricase or glucose oxidase Uric acid or glucose sensor

Table 2

Biomaterials used in development of biosensors by different industries [6,7]

Biomaterials used Type of biosensor Manufacturers/company

Antibody Air pollutants of Candida albicans Universal Sensors, USA

Choline esterase Choline biosensor (immobilized on graphite elec-

trode)

Thorn EMI Simtec Ltd., UK

Cytochrome B2 Artificial electron acceptor, Hexacynoferrate (þ)

II (used with Pt electrode)

Wolverine Medical, USA

Enzyme membrane (glucose, alcohol,

cholesterol etc.)

Immobilized enzyme membranes with O2, NHþ4

and CO2 electrode

Universal Sensors, USA

Glucose oxidase Glucose sensor (immobilized with O2 electrode) Analytical Instruments Co., Japan

Gambro AB, Sweden

Radlkis Electrochemical Instrum., Hungary

Oriental Electric Co. Ltd., Japan

Glucose biosensor (immobilized with H2O2 probe) Fuji Electric Co., Japan

Kyoto Daichi Kagaku, Japan

Omron Toyoba, Japan

Solea-Tacussel, France

Yellow Springs Instruments, Co., USA

Lactate oxidase Lactate sensor YSI Co., USA

Omron Toyoba, Japan

T. brassicae (yeast) Acetic acid, methanol and ethanol (immobilized

with O2 electrode)

Denki Kagaku Keik Ltd., Japan

Uricase Uric acid sensor (immobilized with H2O2 elec-

trode)

Fuji Electric Co., Japan

Xanthine oxidase Fish freshness biosensor (immobilized on polaro-

graphic electrode)

Pegasus Industrial Specialities Ltd., Canada

Yeast cells BOD biosensors Nissin Electric Co. Ltd., Japan

S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316 309

(ii) by �tailoring� enzymes. Microorganisms or wholecells are more active and stable as they remain in their

natural environment. The first successful subcellular

component based sensor has been developed for gluta-

mine measurements in which mitochondrial fraction

containing glutaminase is immobilized at ammonia gas

electrode [10].

The first enzyme electrode, an amperometric type

biosensor, was developed by Clark and Lyons [11]. Theyused a soluble biomaterial glucose oxidase held between

membranes. The oxygen uptake was measured with

oxygen electrode

Glucose þ O2 þ H2O �!Glucose oxidaseH2O2 þ Gluconic acid

Cass et al. reported the use of ferrocene (dicyclo pen-

tadienyl iron) and its derivatives as a mediator in glu-

cose sensor as it transfer the electrons not only

quantitatively but also at faster rates [12]. Marthew et al.

have tested a pen sized glucose meter which uses dis-

posable strips for blood spot application where the fer-rocene technology is employed [13].

Amperometric enzyme electrodes have also been

constructed that facilitate electron transfer between en-

zyme and electrode. Chemical electron transfer media-

tors (ferrocene) are often used, which are substances

that readily exchange electrons with both co-enzyme and

electrode and which therefore open a charge transfer

path from electrode to the enzyme active center [14].Ferrocene is found as good electron transfer mediater as

shown in Fig. 4.

Similarly, another biomaterial penicillinase is widely

used to monitor penicillin content in fermentation

broth. In this type of biosensor, the electrode is based on

use of pH probe coated with immobilized enzyme pen-

icillinase [15]

Penicillin �!PenicillinasePenicilloic acid

It is well known that various chemical compounds suchas amines, aldehydes, ammonia and carbon dioxide are

formed on the meat spoilage process. Several methods

have been used for the determination of meat freshness.

Since various types of amines are produced during meat

spoilage, they can be used as an indicator of freshness.

Karube et al. [16] developed an enzyme electrode con-

sisting of monamine oxidase collagen membrane and

an oxygen electrode for estimation of fish freshness.Monamines formed by microbial action are oxidized to

aldehyde by immobilized enzyme and oxygen con-

sumption is monitored amperometrically by oxygenelectrode. The concentration of monamines is found to

be directly proportional to the difference in current be-

tween initial and final steady state current.

For determination of aspartame (Nutra sweet, also

used by diabetic patients instead of sugar) in several

food and carbohydrate beverages, LL-aspartase enzyme is

used by immobilization on ammonia selective electrode.

LL-aspartame is dipeptide that consists of LL-aspartic acidand LL-phenyl amine i.e. N -LL-aspartyl-LL-phenylamine-

1-methyl ester. An improved sensor was made by co-

immobilization of carboxypeptidase and LL-aspartase on

an ammonia gas sensing electrode [17]. The enzyme

carboxypeptidase A cleaves aspartame to LL-phenyl

amine and LL-aspartic acid

Aspartame �!Carboxypeptidasel-phenylamine

þ l-aspartic acid

The aspartic acid formed is then deaminated by LL-

aspartase with liberation of fumerate and ammonium

ion

l-aspartic acid �!L-aspartaseFumerate þ NHþ

4

The ammonium ions generated were sensed by the am-

monia electrode.

For development of uric acid biosensor, biomaterial

uricase was immobilized onto an amperometric O2 [18]

or H2O2 sensor [19–21] and onto a potentiometric CO2

electrode [22]

Uric acid þ 2H2O þ O2 �!UricaseAllantoin þ H2O2 þ CO2

There are many other enzymes (biomaterials) used in

development of biosensors for specific analytes as shownin Tables 3 and 4.

3. Biomolecules used in microbial biosensors

The use of immobilized microbial cells as biocatalysts

has a number of advantages over both free cells and

immobilized enzymes [27–30]. Due to the high efficiencyof microbial cells, it�s application in one stage and multi-

enzyme processes for production of enzymes, antibio-

tics, co-enzymes, amino acids and carbohydrates has

major advantages [27–29]. Immobilized cells are active

multi-enzyme system regenerating cofactors. Microbial

sensor composed of immobilized yeast Trichosporon

brassicae onto an oxygen electrode for continuous de-

termination of acetic acid in fermentation broths hasbeen developed by Hikuma et al. [31].

Microbial sensor consisting of immobilized whole

cells of Brevibacterium, Lactofermentum [32] and

Pseudomonas fluorescens [33] on an oxygen electrode are

also developed. The determination of sugars (glucose,

2e-

Electrode 2 Ferrocine

GOD(Red)

GOD(Oxi)

2 Ferricinium+ GluconoLactone

Glucose

Fig. 4. Mechanism of action of ferrocine-mediated glucose sensor.

310 S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316

fructose and sucrose) in a fermentation broth for glu-

tamic acid production, total sugars were determined for

the extent of oxygen consumption by the immobilized

microorganisms [32]. Similarly, sensor of glucose, lac-

tate, pyruvate, sucrose and ethanol based on the cells

from the yeast Hansenula anomala are also developed

and the rate of conversion of metabolites determined by

an O2 electrode [34].

Table 3

Enzymes used as biomolecules in development of various type of biosensors and their applications [6,23,24]

Biomaterials (enzymes) Analyte Device/probe Applications

Alcohol oxidase Acetic acid Pt (O2) Blood and saliva alcohol test and fermentation industries

Alcohol oxidase Alcohol Pt (O2), Pt (H2O2) Alcohol test and fermentation industries

Cholesterol oxidase and esterase Cholesterol Pt (H2O2) Cardiovascular diseases

Formate dehydrogenase Formic acid Gas (CO2) Fermentation industry and health care

Glucose oxidase Glucose Gas (O2), Pt (H2O2), Pt

(O2)

Diabetes, fermentation and food industry

Glutaminase Glutamine Cation Myocardial and hepatic diseases

Glutmate dehydrogenase Glutamic acid Cation Myocardial and hepatic diseases

Lactate dehydrogenase Lactic acid Pt[Fe(CN)6] Liver and heart diseases

Lactate oxidase Lactate Pt (H2O2) Human healthcare

Malate dehydrogenase Malate Gas (CO2) Fermentation industry

Nitrate reductase Nitrate NHþ4 Environmental and industrial processes

Nitrite reductase Nitrite NH3 (gas) Environmental and industrial applications

Oxalate oxidase Oxalate Gas (CO2), Pt (H2O2) Diagnosis of hyper-oxaluria in urine (kidney disease)

Penicillinase Penicillin PH Pharmaceutical industry

Succinate dehydrogenase Succinic acid Pt (O2) Fermentation industry

Tyrosine dehydrogenase Tyrosine Gas (CO2) Human healthcare

Urease Urea Gas (NH3,CO2), PH Kidney function test

Uricase Uric acid Pt (O2) Kidney function test

Table 4

Biomolecules (enzymes) sources used in development of biosensors and their applications [25,26]

Biomaterials (enzymes) Available sources Applications

Acetylcholine esterase Electric eel, Bovine erythrocytes Choline sensor for healthcare

Alcohol dehydrogenase Equine liver, Yeast, Thermoanaerobium brockii Alcohol sensor for blood, saliva and fermen-

tation

Alcohol oxidase C. boidinii, H. sp., Pichia pastoris, Hansenula sp. Alcohol sensor for blood and fermentation

process

Cholesterol oxidase Schizophyllum commune, Brevibacterium sp.,

Nocardia erythropolis, P. fluorescens, Streptomyces sp.,

Cellulomonas sp. etc.

Cholesterol sensor for healthcare and food

industry

Cholesterol esterase Bovine pancreas, Porcine pancreas, P. fluorescens Cholesterol sensor for healthcare and food

industry

Cytochrome B2 Heimintho sporium Used in bienzyme lactate sensor (LO/LDH)

Cytochrome C Bovine heart, horse heart, chicken heart, sheep heart,

pigeon breast muscle, rabbit heart, rat heart,

Saccharomyces cerevisiae

Formic acid sensor used in urine, blood and

gastric juices

Glucose oxidase Aspergillus niger Glucose sensor for diabetes

Glutamate decarboxylase Escherichia coli Glutamate sensor for healthcare

Lactate dehydrogenase Lactobacillus leichmannil, Bacillus stearothermophilus,

Staphylococcus epidermidis, rabbit heart, rabbit muscle,

porcine heart and muscle, chicken heart, chicken liver,

bovine heart, trout muscle, human erythrocytes,

Leuconostoc mesenteroides

Lactate sensor for liver and heart diseases

Lactate oxidase Pediococcus sp. Lactate sensor for liver and heart disease

Nitrate reductase E. coli, Aspergillus sp. Nitrate sensor for environment and industrial

application

Urease Jack bean, Cajanus indicus, B. pasteurii Urea sensor for kidney function test and

industrial application

Uricase Porcine liver, Arthobacter globiformis, C. utilis Uric acid sensor for hematology disorder

Xanthine oxidase Butter milk, microorganisms etc. Fish freshness sensor

S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316 311

3.1. Ammonia sensor

Nitrifying bacteria has two genera of bacteria i.e.

Nitrosomonas sp. and Nitrobacter sp. Nitrosomonas sp.

utilizes ammonia as the sole source of energy:

NH3 þ 1=2O2 �!Nitrosomonas sp:NO�

2 þ H2O þ Hþ

Nitrobacter sp. of bacteria oxidize nitrate to nitrite as

follows:

NO�2 þ 1=2O2 �!Nitrobacter sp:

NO�3

The oxidation of both substrates proceeds at high rate

and oxygen consumption by bacteria can be determined

directly by oxygen electrode attached to the immobilized

bacteria. Therefore, ammonia is determined ampero-

metrically by microbial sensor using immobilized bio-

material (nitrifying bacteria) and an oxygen electrode

[35].

3.2. BOD sensor

Biological oxygen demand (BOD) is one of the most

widely used and important indication of organic pollu-

tion. The conventional test method requires 5 days in-

cubation period and includes complicated procedures.

Therefore, rapid and reproducible method was devel-

oped using immobilized yeast (T. cutaneum), sand-wiched between oxygen permitted teflon membrane and

a porous membrane, attached to the oxygen probe�splatinum cathode. When sample solutions were injected

into the system, organic compound was assimilated by

immobilized microorganisms. Consumption of oxygen

by microorganisms caused a decrease in dissolved oxy-

gen using the membrane. As a result, the current of the

sensor decreased with time until a steady state wasreached. The microbial sensor can be used for a long

time for the estimation of BOD [31].

3.3. Fish freshness sensor

Within the period of time between the death of a fish

and its consumption, a large number of biochemical and

physicochemical changes take place. Accurate and rapiddetermination of freshness is essential for the marine

food industry. Various chemical indicators such as vol-

atile basic nitrogen [36], ammonia [37,38], amines [39],

volatile acids [40,41], pH [42–45], adenosine triphos-

phate (ATP) and related compounds [46–58] have been

identified for fish freshness. These all procedures are very

tedious and time consuming. Therefore, it is difficult to

determine fish freshness accuracy by simple indicators.Later, scientists discovered that phosphorylation in an-

imal muscle [46–57], the decomposition of ATP in fish

meat sets in after the death of the fish and ADP, AMP

and related compounds are formed. For determination

of fish freshness, enzyme sensors specific for hypoxan-

thine [59], inosine [60], IMP [61] and AMP [62], have

been developed using immobilized enzyme membrane

and oxygen electrodes. All of these sensors are based on

the principle that the output current of oxygen electrodedecreases due to the consumption when hypoxanthine is

oxidized to uric acid by xanthine oxidase. The phos-

phorylation of fish muscle during decomposition takes

place as shown in Fig. 5.

The simultaneous determination of hypoxanthine and

inosine is also presented in Fig. 5. The oxidation of

hypoxanthine to uric acid by xanthine oxidase causes O2

consumption. A decrease in dissolved O2 around themembrane results and the current from the electrode

decreases. On the other hand, inosine is not affected by

xanthine oxidase and therefore, O2 consumption does

not occur. Hypoxanthine is formed by decomposition of

inosine by enzyme nucleoside phosphorylase comes in

contact with xanthine oxidase membrane as result of

diffusion. Oxygen consumption occur and subsequent

current decreases. Thus, simultaneous determination ofhypoxanthine and inosine is possible for better response.

Nucleoside phosphorylase membrane must be placed

closer to the platinum electrode than xanthine oxidase

membrane (Fig. 6).

Microbial sensor for fish freshness development came

from the observation that during the storage of fish,

high molecular weight components such as protein and

glycogen are gradually degraded into lower molecularweight components, which can be utilized more rapidly

by microorganisms e.g. Altermomonas putrefaciens [63–

67]. Some examples of microorganisms used as bioma-

terial for development of specific biosensor are shown in

Table 5.

ATPase MyokinaseATP ADP AMP

Pi PiAMP deaminase

NH3

IMP

5-Nucleotidase

Pi

Ribose 1-phosphate Nucleosidasephosphorylase

InosineHypoxanthine Pi

O2

Xanthine oxidase

Xanthine oxidaseXanthine Uric acid

O2

Fig. 5. Changes in ATP and related compounds during decomposition

of fish. ATP: adenosine tri phosphate, ADP: adenosine di phosphate,

AMP: adenosine mono phosphate, IMP: inosine mono phosphate.

312 S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316

Cell-based biosensors [68] uses cells as biomaterial for

chemical detection and are composed of two compo-

nents: (1) a cell that expresses a particular receptor

protein immobilized on a prepared substrate and (2) a

transducer setup that records the cell response. Cells aremonitored either by using extracellular electrodes in the

substrate or by using registered photodiode arrays todetect changes in voltage-sensitive dyes in the cell

membrane.

4. Biomolecules used in development of immunosensors

Another possible application of biomaterial is the

construction of immunosensors using antigens or anti-bodies. Immobilized creatine kinase M (CK-M) anti-

body is used as pretreatment for detection of the

cardiospecific CK-MB isoenzyme. Goat antihuman CK-

M Ig G was immobilized on a electrode and that can be

used for several assays and is regenerable [31]. Such type

of sensors have excellent selectivity because of high an-

tibody–antigen specificity. Enzymes are extremely useful

as labels in immunoassays as their catalytic propertiesallow the detection and quantitation of low levels of

immune reactants. The enzymes most commonly used

are alkaline phosphatase, horseradish peroxidase, glu-

cose oxidase and b-galactosidase.

Immunologically based sensors have a three-pronged

approach for the development of immunologically based

optical sensors for environmental, clinical, and defence

applications [73,74]. The first element of the approachfocuses on performing a localized immunoassay on the

+ -

Oxygen electrode

Teflon membrane

Nucleoside phosphorylase membrane

Cellulose membrane

Xanthine oxidase membrane

Fig. 6. Schematic representation of biosensor for fish freshness.

Table 5

Microorganisms used as biomolecules for development of microbial biosensors and their applications [69–72]

Biomaterials (microorganisms) Type of sensor Device/probe Applications

Azotobacter vinelandii Nitrate Gas NH3 Ammonia sensor in industrial process

B. subtilis Mutagenes Gas O2 Screening of mutagens by microbial sensor

Bacterium cadaveris Aspartic acid Gas NH3 Amino acid sensor for healthcare and food

industry

Citrobacter fseundi Formic acid Fuel cell Fermentation industry, culture media, urine,

blood and gastric juices

Clostridium acidurici LL-serine Gas NH3 Amino acid sensor for healthcare indicators

Desulfovibrio desulfuricans Sulfate Sulfide ISE Agriculture industry

E. coli LL-tryptophan Gas NH3 Amino acid sensor

E. coli Lysine CO2 Amino acid sensor for fermentation and food

industry

E. coli Glutamic acid CO2 BOD, healthcare and food industry

E. coli Glutamic acid Gas CO2 BOD, healthcare and fermentation

Enterobacter agglomerans Ascorbic acid Gas O2 Pharmaceutical industry

L. arabinosis Nicotinic acid pH electrode Drug industry

Methylomonas flagelatis Methane O2 Fermentation industry

Nitrifyingbacteria (Nitrosomonas and

Nitrobacter sp.)

Ammonia O2 Clinical and industrial process

N. erythropolis Cholesterol Gas O2 Blood cholesterol and food industry

Proteus morganii LL-cysteine Gas H2S Amino acid sensor

P. fluorescens Glucose O2 Molasses industry

Pseudomonas sp. LL-histidine Gas NH3 BOD in waste water

S. cerevisiae Glucose, sucrose, fruc-

tose

Gas O2, CO2 Food industry and beverages

Sarcina flava Glutamine Gas NH3 Amino acid sensor

Streptococcus faecium LL-arginine Gas NH3 Amino acid sensor

T. brassicae Acetic acid O2 Fermentation industry

T. brassicae Ethanol O2 Fermentation industry

T. cutaneum BOD O2 Environmental pollution monitor

S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316 313

surface of an optical fiber. Changes in fluorescence orchemiluminescence are detected if the analyte is present.

This approach implements advances in fluorescence,

optical, and biochemical technologies to improve the

speed, sensitivity and utility of such optical sensors. The

second approach involves fabricating a continuous flow

immunosensor, which relies on displacing labeled anti-

gen from immobilized antibody in the presence of the

species to be detected. As little as 5 pg TNT can bedetected in less than 1 min. The third approach involves

fabricating arrays of antibodies organized on a dispos-

able chip. The array-based sensor can be interrogated

optically using fluorescence or infrared detectors.

4.1. Ultrasensitive fiber-optic biosensors

Fiber-optic biosensor has been developed for therapid analysis of clinical and environmental samples [75–

77]. Real-time fluoroimmunoassays for multiple agents

require adaptation to the miniaturized instrumentation

and new fluorescent labels. Fiber-optic fluorometers are

used to monitor the binding of antigen to antibody

immobilized near the distal end of a long optical fiber.

Since the measurement occurs at the fiber�s surface in the

evanescent wave, only detection of bound fluorophoresis possible. This improves the speed, sensitivity and

utility of immunoassays [78].

Two basic techniques are used in optic fibre biosen-

sors, total internal reflection spectroscopy (IRS) and

fluorescence spectroscopy via the transmission of both

fluorophore excitation and emission light along one

more fibres and filtering the signal being required to

separate out the wavelength of interest.When a light beam, travels in a transparent and op-

tically dense medium, it will strike the interface with a

less dense medium and total internal reflection (TIR)

occur, provided that incident angle is above a certain

critical angle [79,80]. When light is retained within the

waveguide in this way, not all the energy is confined

within the medium in which the light is being propa-

gated (Fig. 7A).The internally reflected light generate an electro-

magnetic ‘‘evanescent wave’’ which penetrate the lower

density reflecting medium at the point of reflection for a

distance comparable with the wavelength of light. The

evanescent may be used to excite the fluorescent mole-

cule. This provides elimination of washing steps in an

immunoassay.

When surface (interface between two media of dif-ferent refractive index) of the waveguide is coated with

this layer of metal, then evanescent wave can couple

with electron plasma of the metal, causing the electron

to oscillate and generate surface plasmon wave. Most of

the incident light is converted to surface plasmon causes

sharp reduction in the intensity of reflected light due to

the surface plasmon resonance (SPR). The angle at

which SPR is dependent on the surface metal layer

thickness and refractive index of the medium contact

with the layer at the surface opposite to that at which

reflection occurs (Fig. 7B). Therefore, on binding of

antibodies at the metal layer, the refractive index of themedium in contact with the metal will change, resulting

in an alternation of the angle at which resonance occurs.

Similarly, on binding of the antigen to antibody, the

refractive index of the medium will change again and a

second change in the angle at which resonance occurs

will take place (Fig. 7C).

Acknowledgements

The authors are thankful to Prof. S.K. Brahmachari,

Director, Institute of Genomics and Integrative Biology

(formerly: Centre for Biochemical Technology), Delhi,

! ! ! ! ! !

Light

Prism

Metal layer

Sample (Antibody)

Angle ofincidence

! ! ! !100 200 300 400

Time (sec)

Antigenbinding

Antibodybinding

Antibody

Resonancesignal (kRU)

18

22

24

20

θ

Evanescent field

Light intensity

Waveguide

(A)

(B)

(C)

Fig. 7. Basic principle of optical fibre biosensors: (A) generation of

evanescent wave, (B) surface plasma resonance, (C) application of SPR

in immunosensor.

314 S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316

for encouragement in writing this manuscript. SandeepK. Sharma thanks CSIR, New Delhi for providing fel-

lowship.

References

[1] F.S. Ligler, Lecture on Biosensors, June 5, 1997, Biosensor and

Biomedical Laboratory, Naval Research Laboratory, ASEEE-

1818 N Street, NW, Washington DC, 200369 (2000). Available

from <www.asee.org>.

[2] C. Reid, Biosens. Bioelectron., June 5 (2000). Available from

<www.cynthiaReid@worldnet.attnet>.

[3] J.H.T. Luong, A. Mulchandani, G.G. Guilbault, Trends Biotech-

nol. 6 (1988) 310.

[4] J.A. Kas, R.M.J. Brown, C.E. Schmidt, Natural Polym. (2000).

Available from <www.asee.org>.

[5] J. Pickup, Trends Biotechnol. 1 (1993) 285.

[6] G.G. Guilbault, G. de Olivera Neto, Immobilized enzyme

electrode, in: J. Woodward (Ed.), Immobilized Cells and Enzymes:

A Practical Approach, IRL Press, Oxford, 1985, p. 56.

[7] F.W. Scheller, D. Pffiffer, F. Schubert, R. Renneberg, D. Kirstein,

Application of enzyme based amperometric biosensors to the

analysis of real samples, in: A.P.F. Turner, I. Karube, G.S. Wilson

(Eds.), Biosensor––Fundamental and Applications, Oxford Uni-

versity Press, New York, 1987, p. 319.

[8] D.P. Nikolelis, Anal. Chim. Acta 161 (1984) 343.

[9] S.A. Barker, Immobilization od biological components of biosen-

sor, in: A.P.F. Turner, I. Karube, G.S. Wilson (Eds.), Biosensor––

Fundamental and Application, Oxford University Press, New

York, 1987, p. 85.

[10] M.A. Arnold, G.A. Rechnitz, Biosensors based on plant and

animal tissues, in: A.P.F. Turner, I. Karube, G. Wilson (Eds.),

Biosensors––Fundamentals and Applications, Oxford University

Press, New York, 1987, p. 31.

[11] L. Clark, C. Lyons, Ann. N.Y. Acad. Sci. 102 (1962) 29.

[12] A.E.G. Cass, G. Davis, G.D. Francis, Anal. Chem. 56 (1984) 667.

[13] D.R. Marthews, R.R. Holman, E. Brown, Lancet 1 (1987) 778.

[14] J.C. Pickup, Biosensors for diabetic mellitus, in: D.L. Wise (Ed.),

Applied Biosensors, Butterworth Publishers, Stometham, MA,

USA, 1989, p. 234.

[15] G. Guilbault, Handbook of Immobilized Enzymes, Marcel

Dekker, New York, 1984, p. 87.

[16] I. Karube, I. Saitoh, Y. Araki, S. Suzuki, Enzyme Microb.

Technol. 2 (1980) 117.

[17] G.G. Guilbault, G.J. Lubrano, J.M. Kaufimann, G.J. Patriarche,

Anal. Chim. Acta 206 (1988) 369.

[18] G.G. Guilbault, M. Nianjo, Anal. Chem. 46 (1974) 1769.

[19] J. Kulis, M. Pesiekiene, V.S. Laurinavicius, S. Tatikyan, A.

Simonyan, Zh. Anal. Khim. 40 (1985) 2077.

[20] M. Jaenchen, G. Gruenig, K. Berterman, Anal. Lett. 18 (1985)

1799.

[21] H. Iwai, S. Akihama, Chem. Pharm. Bull. 34 (1986) 3471.

[22] T. Kawashima, A. Arima, N. Hatakeyma, N. Tominaga, M.

Ando, Nippon Kaqahu Kaishi (1980) 1542.

[23] S.S. Kuan, G.G. Guilbault, Ion selective electrodes and biosensors

based on ISEs, in: A.P.F. Turner, I. Karube, G.S. Wilson (Eds.),

Biosensor––Fundamental and Applications, Oxford University

Press, New York, 1987, p. 44.

[24] R.R. Coulet, G. Bardeletti, F. Schaud, in: D.L. Wise (Ed.),

Amperometric Enzyme Membrane Electrode, Marcel Dekker,

Bioinstrumentation and Biosensor Inc., New York, 1991, p. 758.

[25] B.J. Gould, B.F. Rocks, Enzyme in clinical analysis––data, in: A.

Wiseman (Ed.), Hand Book of Enzyme Biotechnology, Haisted

Press, New York, 1986, p. 434.

[26] Sigma Biochemicals and reagents for life science research

catalogue, Sigma-Aldrich Co., USA, 2001.

[27] K.A. Koshcheyenko, Prikil. Biochim. Microbiol. 27 (1981) 477.

[28] K.A. Koshcheyenko, Microbiologia 11 (1981) 55.

[29] S. Fukui, A. Tanaka, Annu. Rev. Microbiol. 36 (1982) 45.

[30] F.B. Kolor, Process Biochem. 17 (1982) 12.

[31] M. Hikuma, H. Suzuki, Y. Yasuda, I. Karube, S. Suzuki, Eur.

J. Appl. Microbiol. Biotechnol. 8 (1979) 289.

[32] M. Hikuma, H. Obamna, T. Yasuda, I. Karube, S. Suzuki,

Enzyme Microb. Technol. 2 (1980) 237.

[33] I. Karube, S. Mitsuda, S. Suzuki, Eur. J. Appl. Microbiol.

Biotechnol. 7 (1980) 343.

[34] J. Kulys, K. Kadziauskiene, Biotechnol. Bioeng. 22 (1980) 221.

[35] I. Karube, T. Okada, S. Suzuki, Anal. Chem. 53 (1981) 1852.

[36] A. Takase, Nippon Suisan Gakkaishi 19 (1953) 71.

[37] F. Ota, Z. Oshiro, Nippon Suisan Gakkaishi 19 (1954) 1150.

[38] Y. Yamamura, Nippon Suisan Gakkaishi 2 (1933) 118.

[39] T. Tokunaga, H. Iida, K. Miwa, Nippon Suisan Gakkaishi 43

(1977) 219.

[40] T. Suzuki, Nippon Suisan Gakkaishi 19 (1953) 102.

[41] S. Asakawa, Nippon Suisan Gakkaishi 24 (1959) 714.

[42] T. Kawabata, M. Fujimaki, K. Amano, F. Tomiya, Nippon

Suisan Gakkaishi 18 (1952) 124.

[43] M. Mikaye, K. Hayashi, Nippon Suisan Gakkaishi 21 (1955) 123.

[44] Y. Yamamura, Nippon Suisan Gakkaishi 5 (1936) 98.

[45] M. Yamamoto, M. Sonehara, Nippon Suisan Gakkaishi 19 (1953)

761.

[46] S. Ehira, H. Uchiyama, Nippon Suisan Gakkaishi 35 (1969) 1080.

[47] T. Saito, Nippon Suisan Gakkaishi 27 (1961) 461.

[48] Y. Fujii, H. Uchiyama, S. Ehira, E. Noguchi, Nippon Suisan

Gakkaishi 32 (1966) 410.

[49] S. Ehira, M. Anekawa, Nippon Suisan Gakkaishi 32 (1966) 716.

[50] T. Saito, K. Arai, M. Matsuyoshi, Nippon Suisan Gakkaishi 24

(1959) 749.

[51] H. Uchiyama, S. Ehira, Nippon Suisan Gakkaishi 36 (1970) 977.

[52] K. Yamada, S. Higashino, T. Kawahara, R. Ito, Nippon Suisan

Gakkaishi 47 (1981) 631.

[53] K. Numata, H. Suzuki, K. Usui, Nippon Shokuhin Kogyo

Gakkaishi 28 (1981) 542.

[54] N.R. Jones, J. Murrey, E.I. Livingston, C.K. Murrey, J. Sci.

Food. Agric. 15 (1964) 763.

[55] N.R. Jones, J. Murrey, J.R. Burt, J. Food Sci. 30 (1965) 791.

[56] F.D. Jahns, J.L. Howe, R.J. Coduri, A.G. Rand, Food Technol.

30 (1976) 27.

[57] J.R. Burt, J. Murrey, G.D. Stroud, J. Food Technol. 31 (1968)

165.

[58] E.H. Lee, T. Oshima, C. Koizumi, Nippon Suisan Gakkaishi 48

(1982) 255.

[59] E. Watanbe, K. Ando, I. Karube, H. Matsuoka, S. Suzuki, J.

Food Sci. 48 (1983) 496.

[60] E. Watanbe, K. Toyoma, I. Karube, H. Matsuoka, S. Suzuki,

Appl. Microbiol. Biotechnol. 19 (1984) 18.

[61] E. Watanbe, K. Toyoma, I. Karube, H. Matsuoka, S. Suzuki, J.

Food Sci. 49 (1984) 114.

[62] E. Watanbe, T. Ogura, K. Toyoma, I. Karube, H. Matsuoka, S.

Suzuki, Enzyme Microb. Technol. 6 (1984) 207.

[63] T. Saito, K. Arai, M.A. Matsuyoshi, Nippon Suisan Gakkaishi 24

(1959) 749.

[64] S. Ehira, H. Uchiyana, Nippon Suisan Gakkaishi 35 (1969) 1080.

[65] M. Hikuma, T. Kubo, T. Yasudo, I. Karube, S. Suzuki,

Biotechnol. Bioeng. 21 (1979) 1845.

[66] M. Hikuma, T. Kubo, T. Yasudo, I. Karube, S. Suzuki, Anal.

Chim. Acta 109 (1979) 33.

[67] E. Watanabe, A. Nagumo, M. Hoshi, S. Konagaya, M. Tanaka, J.

Food Sci. 52 (1987) 592.

[68] F.S. Ligler, D.A. Stenger, Fabrication of cell based biosensors,

Centre for Biomedical Science and Engg., Naval Research

S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316 315

Laboratory, ASEEE-1818 N Street, NW, Washington DC, 200369

(2000). Available from <www.asee.org>.

[69] I. Karube, Micro-organism based sensor, in: A.P.F. Turner, I.

Karube, G.S. Wilson (Eds.), Biosensor––Fundamental and Ap-

plications, Oxford University Press, New York, 1987, p. 13.

[70] I. Karube, Micro-organisms based sensors, in: A.P.F. Turner, I.

Karube, G.S. Wilson (Eds.), Biosensors: Fundamental and

Applications, Oxford University Press, Oxford, 1989, p. 28.

[71] L. Macholan, in: D.L. Wise (Ed.), Biocatalytic Membrane

Electrode, Marcel Dekker, Bioinstrumentation and Biosensor

Inc., New York, 1991, p. 349.

[72] I. Karube, S. Suzuki (Eds.), Microbial Biosensors, Biosensors––A

Practical Approach, IRL Press at Oxford University Press, New

York, 1996, p. 165.

[73] F.S. Ligler, G.P. Anderson, D.W. Conard, A.W. Kusterheek,

Immunologically based sensors, Centre for Biomedical Science

and Engg., Naval Research Laboratory, ASEEE-1818 N

Street, NW, Washington DC, 200369 (2000). Available from

<www.asee.org>.

[74] H.J. Kwon, H.I. Balcer, K.A. Kang, Comp. Biochem. Physiol. A.

Mol. Integr. Physiol. 132 (2002) 231.

[75] M.A. Cooper, Nat. Rev. Drug. Discov. 1 (2002) 515.

[76] N.M. Velasco-Garcia, M. Toby, Trends Biotechnol. 19 (2001)

433.

[77] S. Dong, X. Chen, J. Biotechnol. 82 (2002) 303.

[78] G.P. Anderson, F.S. Ligler, L.C. Shriver-lake, Ultrasensitive fiber-

optic biosensors, Centre for Biomedical Science and Engg., Naval

Research Laboratory, ASEEE-1818 N Street, NW, Washington

DC, 200369 (2000). Available from <www.asee.org>.

[79] D. Griffiths, G. Hall, Trends Biotechnol. 1 (1993) 122.

[80] G.A. Robinson, Optical immunosensors, in: A.P.F. Turner (Ed.),

Advances in Biosensors, Jai Press, London, 1991, p. 229.

316 S.K. Sharma et al. / Current Applied Physics 3 (2003) 307–316