progress in enzyme-based biosensors using optical transducers

Microchim. Acta 148, 107–132 (2004)

DOI 10.1007/s00604-004-0273-8

Fundamental Review

Progress in Enzyme-Based Biosensors Using Optical Transducers

Martin M. F. Choi�

Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, P.R. China

Received July 6, 2004; accepted August 24, 2004; published online October 21, 2004

# Springer-Verlag 2004

Abstract. Enzyme-based biosensors are well devel-

oped and relatively mature technique in the biosensing

field. Biosensors that utilise enzymes as the recogni-

tion elements represent the most extensively studied

area. The organisation of an enzyme-based biosensor

requires the integration of the biocatalyst with the sup-

port or immobilised materials to the extent that the

biocatalytic transformation is either optically or elec-

tronically transduced. Any optical or electrical changes

at the support material as a result of the biocatalytic

process, that is, depletion of the reactant or formation of

the product, provide routes for the opto=electronic

transduction of the biological process occurring at

the sensing surface. This review focuses on the dis-

cussion of some enzyme immobilisation techniques

including physical and chemical immobilisation.

Enzyme-based biosensors using various optical detec-

tion methods such as absorptiometry, luminometry,

chemiluminescence, evanescent wave, and surface

plasmon resonance are also included. Finally, different

types of enzyme-based optical biosensors for ascorbic

acid, bilirubin, cholesterol, choline, ethanol, glucose,

glutamate=glutamine, lactate, penicillin, urea, and uric

acid determinations are presented.

Key words: Optical transducers; enzyme; biosensors.

Biosensors and their associated techniques are a fast

and rapid growing field which combine biochemistry,

biology, chemistry, physics, electronics and computer

science, etc. The term ‘‘biosensor’’ began to appear in

the scientific literature in the late 1970s. The first bio-

sensor, known as the ‘‘enzyme electrode’’, was pres-

ented by Clark in 1956 [1], and Clark and Lyons in

1962 [2] when the enzyme glucose oxidase (GOx) was

coupled with an amperometric oxygen (O2) electrode.

The change in the dissolved O2 was sensed by the elec-

trode and shown to be proportional to the concentration

of glucose in the sample. In the years that followed,

enzyme electrodes for a variety of other clinically

important substances were also demonstrated by incor-

porating the relevant enzymes to electrochemical sen-

sors. In 1977, Rechnitz et al. [3] designed a selective

electrode for arginine by immobilising living microor-

ganisms on the surface of an ammonia gas-sensing elec-

trode and they described it as a ‘‘bio-selective sensor’’.

Bio-selective sensor was later shortened to ‘‘biosensor’’

and has remained as the popular term for detection

devices which combines a sensing material of biological

origin and a physical transducer. Since then, the designs

and applications of biosensors in various fields of ana-

lytical science have continued to grow tremendously.

IUPAC [4, 5] recently defines a biosensor as a spe-

cific type of chemical sensor comprising a biological or

biologically derived recognition element either inte-

grated within or intimately associated with a physico-

chemical transducer. The biological element is capable

E-mail: [email protected]�On sabbatical leave at The University of North Carolina at

Chapel Hill in July 2004–July 2005

of recognising the presence, activity or concentration of

a specific analyte in solution. The recognition can be a

binding process for example, an affinity-based biosen-

sor consisting of an antibody, nucleic acid, receptor

protein or cell receptor, and synthetic receptor. The

other class of recognition elements is based on biocata-

lytic reactions, for instances, biosensors derived from

enzymes, microorganisms, tissue slices, and biomimetic

catalysts. The interaction of the recognition element

with a target analyte results in a measurable change in

a solution property, such as depletion of a reactant or

formation of a product. The transducer converts the

change in solution property into a quantifiable electrical

signal. The mode of transduction may be one of several

approaches, including electrochemical, optical, piezo-

electric, magnetic or thermometric transducers.

The fundamental and most important feature of a

biosensor is the construction of the biological recog-

nition element or more specifically the biological

recognition site for the interaction with the target ana-

lyte. These biological materials can be proteins, that is

enzymes [6, 7], receptors [6, 8], antibodies or antigens

[9–11], oligonucleotides or deoxyribonucleic acid

(DNA) fragments [12], or low molecular weight

molecules exhibiting affinity interactions with other

biomaterials such as cofactors, namely, nicotinamide

adenine dinucleotide (NAD) [13], biotin [14–18], or

for example, saccharides exhibiting affinity interac-

tions with lectins [19]. For biosensing, the interactions

between the biological recognition elements and the

analytes should also induce some kinds of signals

which can be interpreted and picked up by signal

transducers based on optical spectroscopy (e.g. ab-

sorption, reflectance, luminescence, optical rotation,

evanescent wave, and surface plasmon resonance),

and electrical transduction (e.g. current, potential,

capacitance, impedance, piezoelectric transduction,

and field effect transistor transduction).

Biosensors that utilise enzymes as the recognition

elements represent the most extensively studied area.

The high specificity of enzyme-substrate interactions,

and the usually high turnover rates of biocatalysts,

open the way to tailor-made sensitive and specific

enzyme-based biosensor devices [20–22]. The orga-

nisation of an enzyme-based biosensor requires the

integration of the biocatalyst with the support or

immobilised materials to the extent that the biocata-

lytic transformation is either optically or electroni-

cally transduced. Any optical or electrical changes

at the support material as a result of the biocatalytic

process, that is, depletion of the reactant or formation

of the product, provide routes for the opto=electronic

transduction of the biological process occurring at the

sensing surface as displayed in Fig. 1.

For example, the biocatalytic conversion of glucose

to gluconic acid at the GOx-immobilised materials

results in biocatalysed O2 depletion, hydrogen perox-

ide (H2O2) and Hþ formation.

D-glucose þ O2 þ H2O

��!glucose oxidaseD-gluconic acid þ H2O2

Therefore, amperometric and chemiluminescence

detection of O2 and H2O2 were often used in the

development of biosensor devices.

The tailoring of biosensing systems requires the

assembly of biomaterials on solid supports and the

design of the appropriate signal transduction between

the biological matrices and the support material. The

chemical means to support the biomaterials include

the immobilisation of enzymes on solid surfaces by

means of polymers [23], membranes [24], carbon

paste [25–28], and sol-gel matrices [29, 30], etc.

Functionalisation of solid surfaces with monolayers

of organic compounds and biomaterials for the attach-

ment of biological recognition elements has been a

subject of extensive research [31]. It is one of the

crucial procedures to produce highly active enzyme-

immobilised materials for biosensing which in fact

can determine the success of a biosensor. Since the

main aim of this article is to review the current prog-

ress of the optical transducers for the enzyme-based

biosensors, the enzyme immobilisation methods can

only be briefly discussed in the later sections. Readers

interested in this topic can go through an excellent

review article [32] to obtain more information.

Optical Enzyme-Based Biosensors

Extensive studies in the field of sensors, and, in par-

ticular, in optical biosensors have been performed

Fig. 1. General scheme of biosensing technique

108 M. M. F. Choi

during the last few decades. The studies were basi-

cally driven by the vital practical needs of industry,

medicine, and environmental control and monitoring,

etc. for such analytical systems and devices. These

needs continue to grow at a rapid pace. Enzyme-based

biosensors using optical transducers have been devel-

oped over nearly half-century. Perhaps the earliest

example of an optical biosensor for clinical applica-

tion is a test strip for glucose in urine commercialised

in 1957 [33]. Glucose oxidase and peroxidase were

coimmobilised onto a cellulose pad. The H2O2 pro-

duced from the enzymatic oxidation of glucose reacts

with o-tolidine in the presence of peroxidase to form a

dye. The colour intensity is proportional to the glu-

cose concentration and determined visually by the

eye. It represents a semiquantitative measurement of

glucose. Later, the colour intensity was determined

more accurately by the reflectance meters.

The success of enzyme-based biosensors is based

on mainly two factors: The first one is the immobili-

sation or fixation of enzymes on solid substrates or

platforms. This step is extremely important as the

immobilisation method can enhance the working life-

time and shelf-time of the biosensors. It also affects

the sensitivity and limit of detection of the biosensors

to analytes. The second part is the optical transducers

which can affect the sensitivity, selectivity and limit

of detection of the biosensors. The sensitivity of a

biosensor depends on the affinity or catalytic proper-

ties of the biological component and the sensitivity of

the physico-chemical transducer. In this article, the

enzyme immobilisation methods are briefly reviewed,

and then followed by various techniques ofoptical trans-

ducers. Finally, some examples of optical enzyme-based

biosensors are presented.

This review covers approximately the period up to

June 2004. Consequently, there is a possibility that

some of the articles will be missed by the time this

review is published. The author has tried to fish out

articles from a computer search of SciFinder Scholar

(CAS). The author apologises for any omissions

which have not caught his eyes. There is certainly

some excellent work on optical enzyme-based biosen-

sors research going on that is not reported here. As

stated above, this review does not claim to be com-

prehensive. Rather, it will offer some guidance in

identifying interesting development in the current

enzyme-based optical biosensors research.

Techniques of Enzyme Immobilisation

The main reason for enzyme immobilisation is to

allow the re-use of immobilised-enzyme over an

extended period of time, hence leading to cost sav-

ings. Furthermore, it allows easier biosensor manipu-

lation and operation. Numerous methods have been

utilised for the immobilisation of enzymes in optical

biosensors and generally the techniques comprise

either chemical or physical methods. Both methods

intend to keep the enzymes on the solid support with-

out being washed out by solutions. Physical immo-

bilisation of enzyme includes adsorption on solid

supports=polymeric gels, entrapment in inorganic=organic polymeric gels, and confinement within semi-

permeable membranes as portrayed in Fig. 2 [34].

Chemical immobilisation of enzymes can be achieved

by covalently bonding the enzymes to functionalised

solid materials or intermolecular cross-linking of the

biomolecules as illustrated in Fig. 3 [34]. In general,

chemical immobilisation can provide longer shelf-life

and stable biosensors; however, the enzymatic activity

often is not as good as in case of immobilised-

enzymes prepared by physical immobilisation.

Physical Immobilisation

In the early stage of development of enzyme-based

optical biosensors, solid enzyme powders or enzyme

solution were deposited onto a solid support and then

kept in position by a semi-permeable membrane. The

semi-permeable membrane allows the free passage of

the substrate and product but confines the enzyme in a

Fig. 2. Physical immobilisation of enzymes

(black circles) by (a) confinement, (b) adsorp-

tion, and (c) entrapment

Progress in Enzyme-Based Biosensors Using Optical Transducers 109

fixed cavity between the membrane and optical trans-

ducer. The selectivity can be maintained by control-

ling the porosity and chemical properties of the

membrane. The simplest and most common of these

semi-permeable membranes are the dialysis mem-

branes, cellulose-based membranes, and ultra-filtra-

tion membranes.

Another type of simple and easy method of physical

immobilisation is the adsorption of enzymes onto solid

supports. The enzyme is dissolved in solution and the

solid material is in contact with the enzyme solution

for a fixed period of time. The unadsorded enzyme is

then removed by washing with buffer. The adsorption

mechanisms are governed by electrostatic attraction,

hydrophobic interaction and van der Waal’s forces. This

method is simple, mild and causes little or no enzyme

inactivation; hence, the enzyme activity is maintained

and the sensitivity of the biosensor will be higher.

Unfortunately, the immobilised enzymes are loosely

bound and tend to desorb from the solid materials after

repeated use. Consequently, this is not the method of

choice for most workers. In order to circumvent this

drawback, various enzymes have been entrapped within

cross-linked organic or inorganic polymers. The prep-

aration is done by cross-linking of the polymer in the

presence of the enzyme; thus, the enzyme can be phy-

sically entrapped in the micro-pores of the polymer.

The size of the pores is small, and this can prevent

larger enzyme from leaching but allows small analytes

to pass through to react with the entrapped enzyme

[34]. Sol-gel methods were popular with the enzyme

adding to the sol-gel solution. After the sol-gel has been

gelled, the enzyme will be entrapped within the poly-

meric network of the porous gel [35].

Chemical Immobilisation

The most popular chemical immobilisation for

enzyme-based biosensors is the covalent bonding of

enzymes to solid supports. The binding of the

enzymes to the solid support is achieved by activating

the surface of the support and following with coupling

to the activated surface. The excess and unreacted

enzymes are removed by washing with buffer. This

technique provides permanent and stable attachment

of enzyme molecules to the support; however, some

of the enzymes are prone to be deactivated in the

coupling reaction. The following section summarises

some common ways of activating the solid surfaces

for the covalent attachment of enzymes.

Cyanuryl chloride (2,4,6-trichlorotriazine) is a

versatile reagent for the activation of surfaces con-

taining hydroxy groups and subsequent immobi-

lisation of enzyme layers [36–38]. Solid surfaces

functionalised by hydroxy groups react with 2,4,6-

trichlorotriazine to oxy-substituted mono- and di-

chlorotriazine layers as shown in Fig. 4 [32]. These

Fig. 3. Chemical immobilisation of enzy-

mes (black circles) by (a) surface covalent

bonding, and (b) cross-linking

Fig. 4. Attachment of enzyme via 2,4,6-trichlorotriazine to solid

surfaces

110 M. M. F. Choi

residues react with the amino acid residues of the

enzymes to form a monolayer of enzyme on the solid

surface.

Similarly, solid surfaces can be functionalised to

carboxylic acid with subsequent attachment with

enzymes. The carboxylic acid residues are converted

to an active ester using carbodimide reagents [39], to

acyl halides using thionyl chloride [40], or to a mixed

anhydride by the reaction with an anhydride [41] prior

to the coupling of the enzymes on the solid support as

displayed in Fig. 5 [32]. Likewise, solid surfaces can

be functionalised to amino groups with subsequent

attachment with enzymes. The enzyme is coupled

with the amino residues surface by glutaraldehyde

[39] as shown in Fig. 6 [32].

Alkoxy- or halosilanes are reactive substances for

the derivatisation of hydroxy-functionalised supports,

and their hydrolysis groups yields siloxane mono-

layers or multilayers on the solid surfaces (Fig. 7

[32]). In essence, the covalently linkage of enzyme

to solid surfaces can be achieved by a wide range of

chemical reactions. Fig. 8a and b [32] lists a series

of functionalities used for the modification of solid

surfaces and applied to the secondary coupling

of enzymes. The amino functional groups of lysine

residues on the enzymes can be linked to surface

carboxy groups generating amide bonds or to surface

aldehyde functional groups to yield imine bonds

[42–44]. Alternative techniques for attaching enzymes

to solid surfaces include nucleophilic substitution of

Fig. 5. Covalent linkage of enzyme layers to solid sur-

faces by coupling of carboxy functionalities

Fig. 6. Covalent linkage of enzyme layers to

solid surfaces by coupling of amino function-

alities


surfaces funtionalised with cyanuric chloride [45–52],

incorporation of the isothiocyanate functionalities

[53, 54], and Michael additions to quinone function-

alities linked to solid surfaces [55]. Aromatic amines

linked to surfaces enable the generation of the sur-

face-bound diazonium salt, which reacts with tyro-

sine or histidine residues to form enzymes linked

to the solid support through azo groups [56]. The

photochemical activation of a phenylazide monolayer

yields the respective nitrene, which reacts with the

amino group of lysine residues of the enzyme [57].

Enzymes containing cysteine residues can be cova-

lently linked to surfaces functionalised by p-mercury-

benzoate [58], iodoacetamide [59] or maleimide

[60–62].

The immobilisation of enzymes by cross-linking

with bifunctional reagents provides another means

for incorporating enzymes onto solid surfaces. The

enzyme molecules can be either cross-linked with

each other or in the presence of a functionally

protein such as bovine serum albumin. The latter

reagent is added in order to increase the number of

cross-linking sites and to provide physical strength.

Of the considerable number of cross-linking agents

available, glutaraldehyde has found widespread use

in enzyme-based optical biosensors. The glutaralde-

hyde cross-linking procedures have been attractive

due to their simplicity and the strong chemical bond-

ing achieved by the biomolecules. The main draw-

back has been the possibility of activity losses due to

the distortion of the active enzyme conformation and

the chemical alterations of the active site during

cross-linking [32].

Optical Transducers

The transducer is an important component in a bio-

sensor through which the measurement of the target

analyte(s) is achieved by selective transformation of

a biomolecule-analyte interaction into a quantifiable

electrical or optical signal. A wide range of optical

and electrochemical instruments have been em-

ployed in conjunction with biosensing. In general the

optical transducers of most common enzyme biosen-

sors is based on optical techniques such as absorp-

tion, reflectance, luminescence, chemiluminescence,

evanescent wave, surface plasmon resonance, and

interferometry.

Absorbance=Reflectance-Based Transducers

The optical detection principle of absorbance trans-

ducers is fundamentally based on the Beer-Lambert

law:

log ðIo=IÞ ¼ A ¼ "Cl

where Io is the intensity of incident light; I is the

intensity of transmitted light; A is absorbance; " is

the molar absorbance of the analyte at a specific

wavelength; C is the concentration of analyte; and l

is the path length of light through solution.

Fig. 7. Assembly of functionalised, layered siloxane assem-

blies on solid surfaces for the immobilisation of enzymes. Z

is a complementary functional group for X

112 M. M. F. Choi

Fig. 8. (a) Various monolayer surface functionalities for the covalent linkage of enzymes. (b) Various monolayer surface functionalities for

the covalent linkage of enzymes


Some biosensing methods employed this photo-

metric properties at which the analyte or product

involve absorbance changes at specific wavelengths.

The common absorbance transducers use a single fibre

or fibre bundle brings light to the analyte-sensitive

reagent phase and the transmitted or reflected light is

Fig. 8 (continued)

114 M. M. F. Choi

returned to a measurement instrument or detector via

fibre(s) as displayed in Fig. 9a. The extent of the ab-

sorption depends on the absorption cross-section of the

transducing molecule, the optical path length and the

illumination wavelength. Changes in chemical envi-

ronment can modify the absorption of the biorecogni-

tion element, and this modification is monitored as a

change in transmitted intensity within the biosensor.

When dealing with non-transparent measuring

environment, it becomes difficult to measure trans-

mitted light satisfactorily and in these cases the inten-

sity of the reflected light may be used as a measure of

the colour of the recognition element, analyte, or prod-

uct as shown in Fig. 9b. The Kubelka-Munk equation

provides an effective approach for quantitatively relat-

ing the observed signal to the sample concentration

for diffuse reflectance measurements. The Kubelka-

Munk function, f(R), is defined as:

f ðRÞ ¼ ð1 � RÞ2=2R

where R is the absolute diffuse reflectance. This func-

tion is directly related to the concentration of an

absorbing sample species, C, by

f ðRÞ ¼ "C=S

where " is the molar absorbance of the sample species

and S is the scattering coefficient of the sample

surface.

The best known example of biosensor is the involve-

ment of NADþ=NADH in the biosensing reactions:

pyruvateþNADHþHþ��!lactate

dehydrogenaseL-lactateþNADþ

NADH has a strong absorbance at absorption peak

maximum 340 nm, but NADþ has no absorbance at

this wavelength. NADH also gives fluorescence at

�450 nm. The absorbance-based transducer can moni-

tor the absorption change at 340–360 nm and the sig-

nal is then related to the concentration of pyruvate or

L-lactate.

Luminescence Transducers

Luminescence transducers combine high selectivity

and sensitivity to monitor changes of various param-

eters in the analytical system such as concentration

of the analytes and products, concentration of quench-

ers, protolytic and complexation reactions, membrane

potential, and hydrophobicity or hydrophilicity, etc.

Luminescence transducers provide a large and im-

portant basis for the development of enzyme-based

biosensors. Main approaches in luminescence sens-

ing utilise either luminescence intensity or lifetime

measurement.

The relationship between luminescence intensity

and analyte concentration under weakly absorbing

solutions can be described by the Parker’s equation:

L ¼ 2:31Io�"Clk

where L is the luminescence intensity; Io is the inten-

sity of excitation light; � is the luminescence quantum

yield; " is the molar absorbance of the analyte at the

excitation wavelength; C is the concentration of ana-

lyte; l is the path length of light through solution; and

k is an instrumental constant.

Fig. 9. Absorbance-based optical transducer with (a) absorption configuration, and (b) reflectance configuration


The construction of luminescence-based trans-

ducer is similar to that of an absorbance-based

transducer. Excitation light is guided to the recogni-

tion element which is exposed to the analyte. The

fluorescence is then collected by the detection sys-

tem as depicted in Fig. 10. Any change in lumines-

cence intensity, phase or lifetime can be related to

the interaction of recognition element, analyte, and

product.

In addition, the principle of luminescence quench-

ing is also commonly employed in luminescence-

based transducer. According to Stern and Volmer,

the relationship between intensities or lifetimes in

the absence and presence of quencher is given by:

Io=I ¼ 1 þ KSV ½Q� ¼ �o=�

where Io and I are the luminescence intensities in the

absence and presence of quencher Q, respectively;

KSV is the Stern-Volmer constant; [Q] is the quench-

er concentration; �o and � are the luminescence life-

times in the absence and presence of quencher Q,

respectively.

A typical example for this type of transducer is

represented by the oxygen sensors based on lumines-

cence quenching of long-lived ruthenium complexes

[63–67]. These oxygen sensors have been applied

successfully to some enzyme-based optical biosensors

which are described in subsequent sections.

Chemiluminescence Transducers

The emission of light due to the chemiluminescence

reaction between reagent and analyte has been em-

ployed in enzyme-based optical biosensors. For

example, Freeman and Seitz [68] developed a H2O2

biosensor based on the peroxidase-mediated oxidation

of luminol by H2O2 at alkaline conditions. In general,

the chemiluminescence reaction can be represented as:

A þ B��!catalystC�

C� ! C þ h�

Chemiluminescence occurs as a result of the

oxidation of certain substances, usually with O2 or

H2O2, to produce light in the cold and in the absence

of any exciting illumination. The optical transducer

is configured to pick up the emitted light and trans-

mitted to an optical detector for recording as shown

Fig. 10. Biosensor with luminescence-based optical transducer. (a) 90�, and (b) 180� arrangement

116 M. M. F. Choi

in Fig. 11. The main disadvantages of this type of

optical transducer are finite lifetime due to reagent

consumption and steady-state mass transfer required

to get a constant signal.

Evanescent Wave Transducer

An interesting approach of optical transducer applied

to enzyme-based biosensors has been proposed. For

example, a biorecognition element is immobilised on

the surface of an optical fibre to sense analyte when it

contacts the fibre surface. At each internal reflection

in the optical fibre, interference between the incident

and reflected internal beam creates a non-propagating

standing wave in the medium, perpendicular to the

reflecting surface. The energy associated with this

wave tails out to the surroundings, where it can inter-

act with analytes in the environment. This tailing phe-

nomenon is known as the evanescent wave. In a rough

estimation, the depth of penetration of the field typi-

cally is in the order of the wavelength of the light

used. Analytes interacted with the recognition element

on the lateral optical fibre surface change the light

propagation characteristics of the optical fibre by their

absorbance, dielectric or reflective index behaviour.

The amount of light leaking into the environment is

easily affected by these changes, and such alterations

can be measured at the distal end of the optical fibre

as depicted in Fig. 12. The major field of evanescent

wave transducers appears to be in immunodetection

with labelled antigen=antibody couples.

Surface Plasmon Resonance Transducer

Another interesting approach for optical transducer

is based on the surface plasmon resonance (SPR)

effect on metal surface. A thin metallic film is

deposited on a polished surface (e.g. a prism) which

acts as the support for the surface plasmon. A high-

ly refractive dielectric biorecognition layer is then

deposited on the metal film to monitor the interac-

tion of biorecognition element and analyte. A plane-

polarised light beam is directed onto the prism

placed on a rotation stage. Reflectance is measured

as a function of angle. The sample solution passes on

the metal surface as shown in Fig. 13. The interac-

tion is highly dependent on the surface refractive

index of the analyte and is detected by the change

in the resonance angle of the exciting plane po-

larised light. Again, the major field of application

of SPR transducer is in the immunosensing, because

the binding of, for example, an antigen on the metal

surface to an antibody in solution causes the reso-

nance angle to change significantly.

Fig. 11. Biosensor with chemiluminescence-based optical transducer

Fig. 12. Biosensor with evanescent wave transducer


Other Optical Transducers

Scientific knowledge will always advance and new opti-

cal techniques will be employed in the enzyme-based

optical biosensors. To date many optical techniques

including interferometry, scattering spectrometry, pho-

toacoustic spectrometry, ellipsometry, polarimetry and

diffraction, etc. have been explored and used as optical

transducers in enzyme-based biosensors. So far their

popularities are not as good as the above-mentioned

optical transducers since they often require complex

and expensive apparatus for construction. Thus, these

optical transducers are not discussed in this review.

However, readers interested with these topics can con-

sult a book edited by Wolfbeis [69].

Examples of Enzyme-Based

Optical Biosensors

Numerous review articles [32, 35, 70–81] have fo-

cused on techniques and theories of enyzme-based

optical and electrochemical biosensors. This review

gives typical and useful examples of some common

enzyme-based optical biosensors. From a practical

and commercial point of view, four typical biosensors

have been widely used. Glucose biosensors are ap-

plied in diagnosis and treatment of diabetes, food

science and biotechnology. Lactate biosensors are

used in sports medicine, critical care, food science

and biotechnology. Urea biosensors are employed in

clinical applications. Glutamate=glutamine biosensors

are utilised in food science and biotechnology. In

addition, other developed enzyme-based optical bio-

sensors such as ascorbic acid, bilirubin, cholesterol,

choline, ethanol, glutamate=glutamine, penicillin, and

uric acid are also presented.

Ascorbic Acid Optical Biosensors

Ascorbic acid is an essential part of healthy diet and

thus it is important to quantify the amount of this

biochemical in food substances. Ascorbic acid is a

reducing agent which can involve in many chemical

reactions and these can form the basis for its detec-

tion. Unfortunately, only very few enzyme-based opti-

cal biosensors for ascorbic acid have been reported.

For instance, an ascorbic acid optical biosensor was

fabricated by immobilising ascorbic acid oxidase on

an oxygen-sensitive sensor membrane which mea-

sured the O2 consumption in the enzymatic oxidation

of ascorbic acid [82]. The detection principle was

simply based on the O2 fluorescence quenching on

an O2-sensitive sensor membrane.

Fig. 13. Biosensor with surface plasmon resonance transducer: (a) experimental arrangement, and (b) attenuated reflective curves for SPR

(1) before and, (2) after exposure to analyte

118 M. M. F. Choi

Bilirubin Optical Biosensors

Bile determination is very useful to diagnose many

gastric pathologies. The determination of bilirubin in

clinical analysis becomes an important issue. Biliru-

bin was oxidised to biliverdin by bilirubin oxidase,

and was quantitatively determined by measuring the

fluorescence quenching of a luminescence dye caused

by O2 consumption in the enzymatic reaction [83, 84].

Alternatively, coupling with immobilized GOx=haemoglobin and glucose, bilirubin was oxidised to

biliverdin by H2O2. The decrease in its absorption at

450 nm was monitored and its concentration was then

determined [85].

Cholesterol Optical Biosensors

The determination of cholesterol levels is of particular

importance in the clinical diagnosis of diseases such

as coronary heart disease, myocardial infarction and

arteriosclerosis. To date most of the enzyme-based

cholesterol biosensors utilise cholesterol oxidase

(ChOx) to catalyse the oxidation of cholesterol by

molecular O2 to 4-cholesten-3-one and H2O2. As a

result, cholesterol can be determined by either mon-

itoring the O2 or H2O2 level optically or ampero-

metrically. For instances, Wu and Choi [86] employed

the hydrogel network matrices to entrap ChOx and

octadecylsilica for optical biosensing cholesterol

in hydrophobic organic or aqueous micelle solvents

in conjunction with an optical O2 transducer. This

organic-phase cholesterol biosensor was successfully

applied to determine the free cholesterol content in

commercial butter samples.

Similarly, other cholesterol biosensors based on

O2 transducer were proposed. Cholesterol oxidase was

entrapped=immobilised in, a cellulose acetate mem-

brane coupled with an O2-sensitive membrane [87], a

graphite powder layer deposited onto an O2-sensitive

silicone film [88], and covalently on a nylon mem-

brane in conjunction with an O2-sensitive membrane

[89].

Alternatively, cholesterol may be determined by

chemiluminescence. Marquette et al. [90] exploited

the luminol electrochemiluminescence for the

development of fibre-optic biosensors for cholesterol.

Zhang and Ma [91] fabricated a fibre-optic biosensor

responding to both of cholesterol esters and free cho-

lesterol by covalently coupling cholesterol esterase,

ChOx and horseradish peroxidase (HRP) to bovine

serum albumin via glutaraldehyde. This chemilumi-

nescence biosensor was based on H2O2-luminol-

HRP to produce the analytical signal.

Choline Optical Biosensors

Choline is an important neurotransmitter in mammals.

Due to the growing needs for on-site clinical monitor-

ing of choline, many efforts have been devoted to

develop choline biosensors. Choline oxidase (CLOx)

and an O2-sensitive dye was dispersed in a Nafion

film. Choline oxidase catalysed the oxidation of cho-

line to betaine and H2O2 while consuming O2.

choline þ 2O2 þ H2O��!CLOxbetaine þ H2O2

The fluorescence intensity of the O2-sensitive dye was

then related to the choline concentration [92]. A simi-

lar approach to determine choline-containing phos-

pholipids in serum with a fibre-optic biosensor was

also described. Choline oxidase was immobilised on

a nylon membrane and positioned onto an O2-sensi-

tive silicone membrane. Phospholipids were hydro-

lysed by the enzyme phospholipase-D to choline

which was subsequently analysed by the O2 trans-

ducer [93]. Furthermore, a fibre-optic biosensor based

on luminol electrochemiluminescence integrated in a

flow injection analysis (FIA) system was developed

for the detection of choline. Choline oxidase was ini-

tially immobilised covalently on solid supports. The

electrochemiluminescence of luminol was generated

by a glassy carbon electrode polarised at þ425 mV vs.

a platinum pseudo-reference electrode to detect

choline [94].

Ethanol Optical Biosensors

Ethanol optical biosensors based on alcohol oxidase

(AOx) and alcohol dehydrogenase (ADH) are com-

monly employed as the ethanol and alcohols recogni-

tion elements. Alcohol oxidase involves the catalytic

oxidation of short-chain aliphatic alcohols whereas

ADH possesses low selectivity for catalytic oxidation

of both aromatic and aliphatic alcohols [95]. Thus,

optical fluorometric biosensors for ethanol was devel-

oped, which was based on the enzymatic oxidation of

ethanol. Sensors layer contained O2-sensitive indi-

cators which showed the decrease in the local O2 par-

tial pressure as the result of the enzymatic oxidation

of ethanol [96, 97]. Other types of optical ethanol

biosensors employed ADH to convert NADþ into


NADH in the presence of ethanol. The rate of produc-

tion of NADH was monitored fluorometrically and

related to the ethanol concentration [98, 99]. Fibre-

optic chemiluminescent biosensors for monitoring

aqueous ethanol also have been fabricated. The bio-

sensors operation schemes were based upon the AOx

enzymatic oxidation of ethanol to produce H2O2,

which then reacted with luminol to produce light.

The intensity of the emitted light was determined

and served as a measure for ethanol concentration

[100–102].

Glucose Optical Biosensors

Continuous monitoring of blood glucose is essential to

avoid the long-term consequences of elevated blood

glucose. A wide variety of optical sensing methods

has been proposed and includes colorimetry [103, 104],

fluorimetry [105–109], absorptiometry [110], near in-

frared spectroscopy [111, 112] and optical rotation

[113, 114]. The most commonly used technology for

blood glucose determination is an enzyme-based

method. Most of these biosensors utilise GOx as the

biological recognition element [115–130]. In the pres-

ence of O2, the enzyme converts glucose into gluco-

nolactaone and H2O2. These biosensors determine the

glucose concentration by monitoring amperometri-

cally the formation of H2O2 or the depletion of O2.

The fabrication of such sensors heavily focuses on

ways to eliminate interferences by, e.g., ascorbate,

urate, and acetaminophen.

For glucose optical biosensors, these interferences

do not exist to a large extent as the optical transducer

does not suffer from these interferents. Instead, the

determination of glucose is done by monitoring opti-

cally either the consumption of O2 or the formation

of H2O2. As O2 is an excellent quencher for most

luminescent compounds, most of the enzyme-based

optical glucose biosensor developed to date is based

on O2 luminescence quenching. Figure 14 displays

the general scheme of this type of glucose optical

biosensors.

In 1984, Uwira et al. [131] reported the first fluori-

metric determination of glucose by measurement of

the depletion in the dissolved O2 in the analyte sample.

The optical transducer was fabricated from an O2-sen-

sitive dye with which the fluorescence intensity was

monitored. Other fibre-optic glucose biosensors have

been prepared by covalently immobilising GOx on

the surface of S-layer ultrafiltration membrane [132],

incorporating GOx in a bilayer lipid film [133],

cross-linking GOx with glutaraldehye [134, 135] and

adsorpting and crosslinking GOx [136] on O2 sensors.

The optical transducers of these biosensors were based

on the luminescence quenching effect of O2 on ruthe-

nium(II) complexes. Other luminescence O2-sensitive

dyes such as decacyclene [137] have also been em-

ployed in O2 transducers for biosensing glucose.

Sol-gel matrices have been proved to be very useful

solid supports for the immobilisation of enzymes as

they can retain the enzyme activity and are considered

to be the best way of immobilising GOx to date [138].

Sol-gel based glucose biosensors employing optical O2

transducers have been developed [35, 139, 140]. The

encapsulated GOx exhibited excellent characteristics

in terms of activity, operational lifetime and optical

transparency. They enabled continuous monitoring of

the glucose concentrations in real samples. Again,

the optical transducers were based on luminescence

quenching effect of O2 on ruthenium(II) complexes.

Eggshell membrane biomaterial has been recently

reported to be an ideal enzyme immobilisation plat-

form for enzymes and the lifetimes of the immobilised

enzymes were much extended. An optical glucose

biosensor was then fabricated from a GOx-immobi-

lised eggshell membrane and an O2-sensitive ruthe-

nium complex sensor membrane [141].

A micrometer-sized fibre-optic fluorescence biosen-

sor for glucose was recently reported [142]. A lumines-

cent O2-sensitive ruthenium complex and GOx were

incorporated into an acrylamide polymer that was

attached covalently to a silanised optical fibre tip sur-

face by photocontrolled polymerisation. The perfor-

mance of this biosensor was comparable with that of

larger glucose biosensors but with the additional advan-

tages of miniaturisation. A disadvantage of this biosen-

sor was that the lifetime was short due to the leaching of

dye and enzyme from the fibre tip after repeating use.

Recently Xu et al. [143] stepped forward to reduce

the size of glucose optical biosensors to nano-scale forFig. 14. General design of glucose optical biosensors

120 M. M. F. Choi

intracellular glucose imaging. These optical nanosen-

sors were fabricated from polyacrylamide PEBBLEs

(Probes Encapsulated By Biologically Localised

Embedding) containing GOx, an O2-sensitive fluores-

cent ruthenium complex, and an O2-insensitive fluo-

rescent reference dye, Oregon Green 488-dextran or

Texas Red-dextran, for the purpose of ratiometric

intensity measurements. The enzymatic oxidation of

glucose to gluconic acid resulted in the local depletion

of O2, which was measured by the O2-sensitive ruthe-

nium dye. The PEBBLES matrix protected the en-

zyme and fluorescence dyes from interference by

proteins in cells. As the size of these nanosensors is

very small, they show potential for intracellular glu-

cose assay.

In addition, Papkovsky et al. [144] described an

immunosensor based on GOx using a luminescence

decay lifetime-based O2 sensor with phase measure-

ments. Although this immunosensor was not intended

to determine glucose, it could be applied to detect

human lactate dehydrogenase isoenzymes. The optical

transducer was operated under luminescence decay

time using the phase measurement technique. Based

on this similar technique, an enzyme-based biosensor

utilising GOx immobilised on a microporous O2 sen-

sor membrane was fabricated to determine glucose by

the phase shift measurement of the luminescent plati-

num complex of octaethylporphine-ketone [145].

The intrinsic fluorescence property of GOx has also

been employed as the optical transducer for glucose

biosensing. The fluorescence from the redox active

site flavine adenine dinucleotides (FADs) of GOx

was monitored on exposure to various glucose con-

centrations [146]. Similarly, Hartnett et al. [147]

focused on the study of the behaviour of GOx seques-

tered within sol-gel derived glasses. The steady-state

and time-resolved fluorescence from FADs of GOx

were monitored when in contact with glucose solu-

tions. The fluorescence intensity decreased with the

increase in glucose concentration. The results demon-

strated that a simple biosensing platform based on the

intrinsic fluorescence from the FAD residues within

GOx could be employed to quantify glucose.

Besides glucose biosensors based on O2 optical

transducers, an optical pH transducer has also been

employed to quantify glucose [148, 149]. Glucose

oxidase was physically immobilised on an optical

fibre. When GOx catalysed the oxidation of glucose

to produce gluconic acid, which, in turn, lowered the

pH in the microenvironment of the biosensor and this

change was then picked up by a pH-sensitive dye

incorporated into the sensing layer.

Finally, Wang et al. [150] reported the electro-

chemiluminescent detection of H2O2 which could be

applied to determine glucose by coimmobilisation of

polymeric luminol, iron(II) tris(5-aminophenanthro-

line) and GOx on an indium tin oxide glass. Unfor-

tunately it was an irreversible sensing scheme as

luminol was consumed during the reactions. Recently

Wolfbeis et al. [151] reported the use of a reversible

H2O2 sensor to quantify glucose by incorporating with

the GOx enzymatic reaction to generate H2O2.

In conclusion, glucose biosensors are, by far, the

most widely employed and therefore continue to drive

research toward better biosensors. The analytical per-

formance of most GOx-based optical biosensors to

date is thus summarised in Table 1 for the ease of

comparison.

Glutamate=Glutamine Optical Biosensors

The amino acid glutamate is the major excitatory neu-

rotransmitter used in the nervous system for interneuro-

nal communication. It is used throughout the brain by

various neuronal pathways including those involved in

learning and memory, locomotion, and sensory percep-

tion. Since glutamate is released from neurons on a

millisecond time scale into sub-micrometer spaces,

the development of a glutamate biosensor with high

temporal and spatial resolution is of great interest for

the study of neurological function and disease. Several

optical biosensors have been developed to monitor glu-

tamate. Glutamate dehydrogenase (GDH) was encap-

sulated in a silica sol-gel film on the tip of an optical

fibre whereas it catalysed the oxidative deamination

of glutamate to �-ketoglutarate and the simultaneous

reduction of NADþ to NADH, whose fluorescence

formed the basis of the detection [152]. Wang and

Arnold [153] employed a dual-enzyme of immobilised

GDH and glutamate-pyruvate transaminase (GPT) to

produce NADH at the tip of a fibre-optic probe. NADH

luminescence was monitored through this probe and

the measured fluorescence intensity was related to

the concentration of glutamate. Glutamate dehydroge-

nase catalyses the formation of NADH, and GPT drives

the GDH reaction by removing the reaction product

and regenerating glutamate. Cordek et al. [154] used

similar methods to immobilise GDH on a submicro-

metre optical fibre and studied the subcellular level

neurophysiological responses of glutamate.


Table

1.

An

aly

tica

lp

erfo

rman

ceo

fvar

iou

sG

Ox

-bas

edo

pti

cal

bio

sen

sors

Bio

sen

sor

des

crip

tio

nT

ran

sdu

cer

Sta

bil

ity

Dy

nam

ic=

lin

ear

ran

ge

Det

ecti

on

lim

it

Res

po

nse

tim

e

Inte

rfer

ence

sR

ef.

En

zym

em

emb

ran

es

wit

h2

-Dcr

yst

alli

ne

stru

ctu

re

O2

lum

ines

cen

ce

qu

ench

ing

20

0h

(co

nti

nu

ou

s

op

erat

ion

)

1–

80

mM

(flow

mo

de)

0.5

–1

5m

M

(ste

ady

-sta

tem

od

e)

?1

00

s?

13

2

Bil

ayer

lip

iden

zym

e

film

O2

lum

ines

cen

ce

qu

ench

ing

?2

–5

mM

??

?1

33

GO

xcr

oss

lin

kin

g

enzy

me

lay

er

O2

lum

ines

cen

ce

qu

ench

ing

40

0h

(co

nti

nu

ou

s

op

erat

ion

)

0.1

–5

00

mM

(FIA

,zo

ne

sam

pli

ng

)

0.1

–1

0m

M

(FIA

,d

irec

tin

ject

ion

)

??

?1

34

GO

x-g

luta

rald

ehy

de

cro

ssli

nk

ing

enzy

me

lay

er

O2

lum

ines

cen

ce

qu

ench

ing

4w

eek

s

(use

dd

aily

)

0.0

6–

1m

M?

6m

in?

13

5

GO

xad

sorp

tio

n

and

cro

ssli

nk

ing

enzy

me

lay

er

O2

lum

ines

cen

ce

qu

ench

ing

6m

on

ths

0–

2m

M?

12

s?

13

6

GO

xim

mo

bil

ised

on

ny

lon

6–

6m

emb

ran

e

O2

lum

ines

cen

ce

qu

ench

ing

5m

on

ths

0.1

–2

0m

M0

.05

–0

.1m

M1

50

–2

10

s?

13

7

En

trap

ped

GO

x

sol-

gel

pow

der

O2

lum

ines

cen

ce

qu

ench

ing

28

0d

ays

0.0

6–

30

mM

6mM

5–

8m

inas

corb

ate,

caff

ein

e

(sli

gh

tin

terf

eren

ce)

13

9

San

dw

ich

edG

Ox

0.1

–1

5m

M2

50

–6

00

s

GO

xen

trap

ped

sol-

gel

O2

lum

ines

cen

ce

qu

ench

ing

>4

mo

nth

s0

.1–

8m

M?

50

–1

00

s?

35

En

trap

ped

GO

x

sol-

gel

pow

der

0.1

–4

mM

15

0–

25

0s

En

trap

ped

-GO

x

insi

lica

texer

og

el

hy

bri

dis

ed

wit

hh

yd

rox

yet

hy

l

carb

ox

ym

eth

yl

cell

ulo

se

O2

lum

ines

cen

ce

qu

ench

ing

3y

ears

0.0

09

–1

00

mM

9mM

6–

9m

inn

14

0

GO

x-i

mm

ob

ilis

ed

egg

shel

lm

emb

ran

e

O2

lum

ines

cen

ce

qu

ench

ing

>5

mo

nth

s0

.3–

2m

M0

.3m

M5

min

asco

rbat

e,u

rate

,

sucr

ose

(sli

gh

tin

terf

eren

ce)

14

1

Mic

rom

eter

-siz

e

op

tica

lfi

bre

glu

cose

bio

sen

sor

O2

lum

ines

cen

ce

qu

ench

ing

4–

6d

ays

0.7

–1

0m

M1

fmo

l;

0.7

5m

M

2s

?1

42

PE

BB

LE

sn

ano

sen

sor

O2

lum

ines

cen

ce

qu

ench

ing

?�

0.3

–8

mM

?�

10

0–

20

0s

n1

43

GO

xo

nm

icro

po

rou

s

lig

ht-

scat

teri

ng

sup

po

rtm

ater

ial

O2

lum

ines

cen

ce

qu

ench

ing

>3

mo

nth

s0

.2–

20

mM

?fe

wse

con

ds

?1

45

122 M. M. F. Choi

Intr

insi

cfl

uo

resc

ence

of

GO

x

FA

Dfl

uo

resc

ence

?1

.5–

2m

M?

2–

30

min

?1

46

GO

xw

ith

inso

l-g

el

der

ived

gla

ss

O2

lum

ines

cen

ce

qu

ench

ing

>6

mo

nth

s�

2–

7m

M?

1–

3m

in?

14

7

Ph

ysi

call

yim

mo

bil

ised

GO

xo

no

pti

cal

fib

re

pH

lum

ines

cen

ce

inte

nsi

ty

?0

.1–

2m

M?

8–

12

min

?1

48

Ph

ysi

call

yim

mo

bil

ised

GO

xo

no

pti

cal

fib

re

pH

lum

ines

cen

ce

inte

nsi

ty

?0

.1–

2m

M?

??

14

9

Po

lym

eric

lum

ino

l,

Fe(

ph

en-N

H2) 3

2þ

,

GO

xse

nso

r

H2O

2el

ectr

och

emi-

lum

ines

cen

ce

?1

0mM

–1

mM

50mM

?as

corb

ate

15

0

GO

xim

mo

bil

ised

on

H2O

2-s

ensi

tiv

e

hy

dro

gel

mem

bra

ne

H2O

2lu

min

esce

nce

tran

sdu

cer

?0

.1–

5m

M�

0.2

mM

>1

0m

inC

u2þ

,F

e3þ

,

citr

ate,

ph

osp

hat

e

15

1

nN

o,?:

no

tav

aila

ble

or

un

clea

r.


Besides GDH, enzymes such as glutamate oxidase

(GLOx) and glutamate decarboxylase (GDC) have

also been used. Kar and Arnold [155] immobilised

GLOx at the tip of an ammonia sensor to produce a

glutamate biosensor whereas Dremel et al. [156] pro-

posed two kinds of fibre-optic glutamate biosensors.

In the first type, an O2 sensor was covered with a

membrane onto which was immobilised GLOx. The

decrease in O2 partial pressure in the presence of glu-

tamate as a result of enzymatic reaction was deter-

mined via dynamic quenching of the fluorescence of

the O2-sensitive indicator. In the second type, a carbon

dioxide-sensitive sensor was covered with a mem-

brane of immobilised GDC. The production of carbon

dioxide in the presence of substrate was determined

via the changes in the pH of a carbon dioxide sensor

consisting of a membrane-covered pH-sensitive fluo-

rescent pH indicator entrapped in a hydrogen car-

bonate buffer. The application of both biosensors to

determine glutamate in food and pharmaceutical sam-

ples was demonstrated.

Glutamine is a major source of nitrogen and carbon

in cell culture media. Thus, glutamine monitoring is

important in bioprocess control. Cattaneo et al.

[157, 158] developed chemiluminescence fibre-optic

biosensor systems for the determination of glutamine

in mammalian cell cultures. Glutaminase (GLA) and

GLOx were immobilised onto porous aminopropyl

glass beads by glutaraldehyde activation and packed

to form an enzyme column. These two enzymes acted

in sequence on glutamine to produce H2O2, which was

then reacted with luminol in the presence of ferricya-

nide to produce a chemiluminescent light signal that

was detected and quantified with a fibre-optic system.

In a similar fashion, glutamate and glutamine were

determined by luminol chemiluminescence with FIA

based on immobilised GLOx and GLA coupled with

Arthromyces ramosus peroxidase. The H2O2 produced

in the enzymatic reactions was determined by luminol

chemiluminescence catalysed by the peroxidase [159].

Lactate Optical Biosensors

Determination of lactate is essential in clinical analy-

sis for the diagnosis of lactate acidosis as a result of

metabolic, respiratory, or haemodynamic disturbance.

Typical fields of application for lactate biosensor are

in sports medicine for exercise control and in food

industry for the control of dairy products. Lactate

oxidase (LOx) is widely employed in biosensors to

determine lactate concentration based on the follow-

ing enzymatic reaction:

CH3CHðOHÞCO2�

ðL-lactateÞþO2��!

LOxCH3COCO2

�

ðpyruvateÞþH2O2

In most lactate biosensors, the consumption of O2 or

the production of H2O2 is monitored amperometri-

cally or optically and the lactate concentration is then

calculated. Other enzymes such as lactate dehy-

drogenase (LDH) and lactate monooxygenase (LMO)

have also been applied in the lactate biosensors:

CH3CHðOHÞCO2� þ NADþ ��!LDH

CH3COCO2�

þ NADH þ Hþ

CH3CHðOHÞCO2�þO2��!

LMOCH3COO�þCO2þH2O

Amperometric lactate biosensors have been studied

extensively as their configuration and design are very

similar to amperometric glucose biosensors. However,

the development of lactate optical biosensor is relative-

ly very scarce. For examples, lactate monooxygenase

was immobilised covalently on nylon membranes,

and the consumption of O2 was measured by follow-

ing, via a fibre-optic bundle, the changes in the fluo-

rescence of an O2-sensitive dye when the biosensor

was in contact with lactate [160]. Another biosensor

for the determination of L-lactic acid based on a fibre-

optic O2 sensor immobilised with LOx was developed.

The consumption of O2 was determined by dynamic

quenching of a fluorescence dye by molecular O2. For

the detection of L-lactic acid in milk products, sample

pretreatment proved necessary for protection of the bio-

sensor. Continuous L-lactate analyses could be carried

out with this enzyme-based biosensor for �2 days

[161]. Similarly, a fibre-optic biosensor based on bacte-

rial cytoplasmicmembranesas the biological recognition

element and an O2-sensitive dye layer as the transducer

has been described for the detection of lactate. Bacteria

with an induced LOx system were adsorbed onto a cel-

lulose disk. The disk was fixed mechanically over the

O2-sensitive siloxane layer on the distal end of an optical

fibre. This system detected lactate with no interference

from glucose, fructose or glutamic acid [162]. Lactate

biosensor based on measurement of phosphorescence

decay time of platinum(II) and palladium(II) complexes

of porphyrins was reviewed by Papkovsky et al. [163].

A fibre-optic biosensor based on the intrinsic fluo-

rescence of immobilised flavoproteins of LMO has been

employed to detect lactate. This method was based on

124 M. M. F. Choi

the change in fluorescence intensity of FAD as a pros-

thetic group of LMO during its interaction with lactate.

The fluorescence was monitored via fibre-optic light

guides at wavelengths >500 nm with fluorescence

excitation at around 410–450 nm. A characteristic fea-

ture of this biosensor is the narrow dynamic range that

usually does not exceed 3 mM [164].

An optical biosensor for the determination of L-

lactate based on immobilised HRP and LOx has been

described [165]. The fluorescence intensity of the

dimeric product of the enzyme catalysed oxidation

of homovanillic acid was utilised to determine H2O2

which was generated from the enzymatic oxidation of

L-lactate in the presence of O2. The biosensor has

been applied to the determination of lactate in control

serum.

Sol-gel encapsulation of LDH and its cofactor has

been employed as a disposable biosensor for L-lactate.

This biosensor utilised the changes in fluorescence

from the reduced cofactor NADH upon exposure

to L-lactate [166, 167]. Although problems such as

diminished enzymatic activity and=or leaching of

enzyme from the sol-gel matrix occurred, the sol-gel

process was sufficiently mild to permit retention of

enzymatic activity. The biosensor had a linear dynamic

range over the normal physiological L-lactate level and

had a long-term storage stability of at least 3 weeks

[149]. An ultrasensitive optical fibre lactate sensor with

rapid response time and 50mm size was developed.

Lactate dehydrogenase was covalently immobilised

onto an optical fibre probe surface and was applied

to determine the lactate content in food samples [168].

A bioluminescent fibre-optic sensor for the analysis

of L-lactate was developed using three different

enzymes, luciferase, NAD(P)H:FMN oxidoreductase

and LDH, immobilised on polyamide membranes.

Two kinds of sensing layer were studied, one consisting

of only one membrane on which the enzymes were

randomly coimmobilised, the other being a compart-

mentalised system obtained by stacking a luciferase=

oxidoreductase membrane on a LDH membrane.

After optimisation, the performances of the biosensor

in terms of sensitivity, detection limit and dynamic lin-

ear range of measurements were strongly improved by

compartmentalisation of the sensing layer compared

with those obtained using the coimmobilised system.

A fivefold increase in biosensor sensitivity was obtained

and the detection limit was 0.2mM lactate [169].

The luminol electrochemiluminescence has been

exploited for the development of several biosensors

allowing the detection of H2O2 and of substrates of

H2O2-producing oxidases. A glassy carbon electrode

was polarised at a fixed potential. Luminol was then

electrochemically oxidised and reacted in the pres-

ence of H2O2 to produce light. Lactate oxidase was

chemically immobilised on collagen or polyamide

membranes. Electro-optical flow injection analysis

of lactate was made possible [90, 170, 171].

Penicillin Optical Biosensors

Determination of penicillin is an important stage in

the control of biotechnological production processes

as well as in the quality control antibiotics prepara-

tion. In most of the developed enzyme-based optical

penicillin biosensors, penicillinase was used to catal-

yse the reaction of hydrolysis of the �-lactam ring in

penicillin to penicilloic acid [172] and the change in

pH was picked up by various pH optical sensors

[173, 174]. For instances, Polster et al. [175] em-

ployed phenol red as the basis of pH measurement.

Healey et al. [176, 177] immobilised penicillinase

and a pH-sensitive dye, fluorescein, on optical im-

aging fibres for fluorometric determination of peni-

cillin. Xie et al. [178] coimmobilised fluorescein

isothiocyanate and penicillinase on a preactivated

biodyne B membrane attached to the end of a bifur-

cated optical fibre for sensing penicillin. In addition,

Carlyon et al. [179] designed a single mode fibre-

optic evanescent penicillin biosensor. Penicillin G

was monitored at 633 nm by the decoloration of the

starch-iodine reagent when Bacillus cereus penicil-

linase was immobilised over the exposed core of a

monomode fibre.

Urea Optical Biosensors

The first enzyme-based urea optical biosensor was

developed by Goldfinch and Lowe [180] in 1984. A

pH-sensitive dye, bromothymol blue, and enzyme

urease were covalently immobilised on a glutathione

membrane. Enzymatic hydrolysis of urea resulted in

an increase in the pH of the medium,

ðNH2Þ2CO þ H2O��!urease2NH3 þ CO2

thus causing the colour change of the pH probe.

Most of the urea optical biosensors developed after-

wards fundamentally employed this enzymatic reaction

and a pH=NH3 optical transducer for determination of

urea. For instances, cellulose acetate membranes with


brilliant yellow [181] or bromothymol blue [182], and

triacetylcellulose membranes with immobilised pH

indicator, neutral red, and urease [183] have been used

as absorption-based transducers for determination of

urea concentration whereas Koncki et al. [184, 185]

immobilised the pH-sensitive polymer polypyrrol=Prussian Blue, and urease on transparent film for spec-

trophotometric measurement of urea.

Another urea optical biosensor relied on the urease-

catalysed hydrolysis product, NH4þ which was

detected with an ion-selective sensing membrane

containing nonactin as ion-selective ionophore and

ETH 5294 chromoionophore in a 1-mm plasticised

poly(vinyl chloride) film. The transducer membrane

and the enzyme containing reaction layer were

sandwich-cast with spin coating onto the surface of

the sensing slide. The attenuation of the laser light

propagating inside the glass waveguide was used as

signal for urea analysis [186].

Wolfbeis and Li [187], and Chen and Wang [188]

designed fluorometric urea biosensors based on an

NH4þ-selective membrane. A urease enzyme layer

was immobilised on the NH4þ-selective polymer

membrane. The NH4þ-selective membrane utilised

dichlorofluorescein octadecyl ester as the anionic

chromophore and nonactin as the neutral ionophore.

Enzymatic hydrolysis of urea produced NH4þ ion

which was extracted into the polymer film to form

complexes with nonactin. Hþ was released which

resulted in a fluorescence change of the sensor mem-

brane due to the charge neutrality principle. This bio-

sensor was applied to determine urea in diluted serum

samples.

De Marcos et al. [189] directly photoimmobilised

urease with polyacrylamide onto a polypyrrole (PPy)

film. This PPy film showed an absorbance spectrum in

the near infrared range which was pH dependent. The

variation of absorbance was thus directly related to the

change of pH caused during the enzymatic reaction,

which was also dependent on the urea concentration.

The main advantage of this biosensor was that no pH-

sensitive indicator was needed.

Covalent immobilisation of amphiphilic mono-

layers containing urease onto optical fibres for fluoro-

metric detection of urea has been proposed [190].

Bifunctional 11 or 12 carbon chain length amphi-

philes with a triethoxychlorosilane group at one

terminus and an amine functionality at the other

terminus were covalently immobilised onto planar

quartz wafers and optical fibres. A small amount of

the fluorescent probe nitrobenzaoxadiazole dipalmi-

toylphosphatidylethanolamine (NBD-PE) was par-

titioned into the membranes from an aqueous

suspension. For coated wafers or fibres placed into

aqueous solutions, alterations of pH change the phys-

ical and electrostatic structure of the membranes,

which in turn altered the emission intensity of the

NBD-PE owing to changes in self-quenching. The

fluorescence intensity decreased as the degree of ioni-

sation of headgroups within the membrane decreased,

consistent with an increase in self-quenching. Urease

was covalently linked onto the functional groups at

the surface of these membranes. Addition of urea to

this system produced NH3 and carbonic acid and

results in changes in fluorescence intensity from the

immobilised layer owing to alteration of surface

charge at the membrane.

Furthermore, Rhines and Arnold [191] employed

two fluoresceins to probe the pH change after the

enzymatic hydrolysis of urea while Xie et al. [192]

developed fibre-optic urea biosensor based on immo-

bilised urease coupled to a fluorescence pH indicator

trisodium 8-hydroxypyrene-1,3,6-trisulphonate. The

enzymatically generated NH3 diffused through the

membrane into a solution of trisodium 8-hydroxy-

pyrene-1,3,6-trisulphonate resulting in the change in

fluorescence intensity. Instead of using absorption and

fluorescence spectroscopies, Gauglitz and Reichert

[193] also proposed the use of reflectance spectros-

copy and a pH-sensitive dye for determination of

urea.

Uric Acid Optical Biosensors

Uric acid and other oxypurines are the principal final

products of purine metabolism in the human body.

Abnormal levels of uric acid can cause gout, hyper-

uricemia, and Lesch-Nyan disease [194]. As such, it

is important to determine the concentration of uric

acid in human urine and=or blood to diagnose these

diseases. A fibre-optic biosensor for uric acid based

on immobilised uricase has been reported [195].

Uricase and HRP were coimmobilised on the inert

matrix bovine albumin via glutaraldehyde. Enzymat-

ic oxidation of uric acid resulted in the production

of H2O2 which subsequently reacted with thiamine

in the presence of HRP to produce the fluorescent

product thiochrome. The fluorescence intensity was

related to the uric acid concentration. Alternative-

ly, a chemiluminescent technique [196] was also

126 M. M. F. Choi

Table 2. Enzyme-based biosensors using optical transducers

Biosensor type Enzyme used Transducer Ref.

Ascorbic acid AsAOx O2-sensitive

indicator=luminescence

intensity quenching

82

Bilirubin BOx O2-sensitive


intensity quenching

83, 84

Cholesterol ChOx O2-sensitive


intensity quenching

86, 88, 89

O2-sensitive


lifetime=phase shift

87

luminol-H2O2

electrochemiluminescence

90

ChOx-HRP luminol-H2O2

chemiluminescence

91

Choline CLOx O2-sensitive


intensity quenching

92, 93

luminol-H2O2


94

Ethanol AOx O2-sensitive


intensity quenching

96, 97

ADH NADH luminescence

intensity

98, 99

AOx luminol-H2O2

chemiluminescence

100–102

Glucose GOx O2-sensitive


intensity quenching

35, 131–137,

139–143

O2-sensitive



144, 145

FAD intrinsic

luminescence intensity

146

pH-sensitive


intensity

148, 149

Fe(phen-NH2)32þ-

luminol-H2O2


150

H2O2-sensitive


intensity enhancement

151

Glutamate GDH-GPT NADH luminescence

intensity

152–154

GLOx pH-sensitive indicator=fluorescence intensity

155

GLOx O2-sensitive


intensity quenching

156

GDH pH-sensitive

indicator=fluorescence

156

Glutamine GLA-GLOx ferricyanide-luminol-H2O2

chemiluminescence

157, 158

GLA-GLOx-

peroxidase

luminol-H2O2

chemiluminescence

159

(continued)


employed to fabricate an uric acid biosensor. Uri-

case and HRP were coimmobilised on a solid sup-

port. Uricase catalysed the oxidation of uric acid to

produce H2O2, which then reacted with luminol

under the catalysis of HRP to produce chemilumi-

nescence. The light intensity was related to the con-

centration of uric acid in the sample solution.

Finally, Table 2 summarises all the above-mentioned

enzyme-based biosensors using various enzymes and

optical transducers.

Table 2 (continued)

Biosensor type Enzyme used Transducer Ref.

Lactate LOx O2-sensitive


intensity quenching

161, 162

LOx O2-sensitive



163

LOx luminol-H2O2


90, 170, 171

LOx-HRP H2O2-sensitive


intensity

165

LMO O2-sensitive


intensity quenching

160

LMO FAD intrinsic

luminescence intensity

164

LDH NADH luminescence

intensity

149, 166–168

LDH, luciferase,

NAD(P)H:FMN

oxidoreductase

bioluminescence 169

Penicillin PC pH-sensitive

indicator=absorption

175

pH-sensitive


intensity

176–178

pH-sensitive

indicator=evanescent

wave absorption

179

Urea UA pH-sensitive

indicator=absorption

180–185

polypyrrole

film=absorption

189

pH-sensitive

indicator=reflectance

193

pH-sensitive


intensity

191, 192

NH4þ-sensitive

film=absorption

186

NH4þ-sensitive

film=fluorescence

intensity

187, 188, 190

Uric acid UC fluorescence intensity 195

luminol-H2O2

chemiluminescence

196

ADH Alcohol dehydrogenase; AOx alcohol oxidase; AsAOx ascorbic acid oxidase; BOx bilirubin oxidase; ChOx cholesterol oxidase; CLOx

choline oxidase; GDC glutamate decarboxylase; GLA glutaminase; GOx glucose oxidase; GDH glutamate dehydrogenase; GLOx glutamate

oxidase; GPT glutamate-pyruvate transaminase; HRP horseradish peroxidase; LDH lactate dehydrogenase; LMO lactate monooxygenase;

LOx lactate oxidase; NADH reduced nicotinamide adenine dinucleotide; PC penicillinase; UA urease; and UC uricase.

128 M. M. F. Choi

Techniques of Background Correction

for Enzyme-Based Optical Biosensors

The success in fabrication of enzyme-based optical

biosensors has been addressed in the previous sec-

tions. Unfortunately, it is often observed that the vari-

able background levels of factors such as dissolved

O2 and pH in real samples can affect the results of

enzyme-based biosensing especially when O2, pH,

NH3, or CO2 sensors are employed as the optical

transducers. Currently several strategies have been

used to overcome this problem [34]. The first method

is to employ a two-sensor technique [35, 148]. For

example, Wolfbeis et al. [35] utilised this technique

to determine the glucose concentration with the first

sensor only sensitive to the dissolved O2 and the other

to both O2 and glucose in the sample solution. The

second strategy is to make use of two different dyes

for ratiometric measurement. For instance, the first

dye serving as a reference indicator is insensitive to

the biosensing process while another indicator is sen-

sitive to the analyte of interest. The ratio of the two

signals is then correlated to the analyte concentration

[143, 197]. Finally the commonly used technique is to

keep the dissolved O2 content and pH of the standards

and samples almost constant by using air-saturated

buffered solutions and performing the analysis in a

relatively closed environment such as continuous-flow

or flow-injection systems [82, 134, 135, 137, 139,

140, 156].

Conclusion and Perspectives

The key elements in the integration of enzymes with

optical transducers include the physical and chemical

deposition of the enzymes on the solid supports, the

electrical=optical communication between the enzyme-

immobilised layers and the transducers, and the elec-

tronic transduction of the biocatalysed transformation

that occurs at the immobilised enzyme=transducer

interfaces. Monolayer and multilayer arrays of en-

zymes can be organised on transducers using co-

valent bonds, affinity interactions, or hydrophobic=hydrophilic interactions. Clearly, the architecture of

enzyme-immobilised layers on optical transducer ele-

ments is envisaged to offer exciting perspectives at the

frontiers of chemistry, biology, physics, medicine, and

material science. Biosensors are one of the most fruit-

ful, exciting, and interdisciplinary areas of research in

analytical science. The concepts of selectivity, revers-

ibility, detection limits, ruggedness, and shelf-life

remain extremely important characteristics of any bio-

sensors and always should be borne in mind. Despite

the complexity of sample matrix and the problems

associated with real sample measurements, significant

progress has already been made in improving reliabil-

ity and extending capabilities to higher sensitivity and

selectivity, and faster response time of biosensors. The

author believes that the biosensor field continues to

advance at an accelerated pace. Looking forward, the

largest market for biosensors is still in clinical and

environmental analysis.

Finally, the current trend of reducing costs of

biological recognition elements and instrumentation

will open new and cost-effective analytical horizons

and broaden existing areas such as clinical and

environmental analysis. Enzyme-based optical bio-

sensors provide novel ways of performing the rapid,

remote, in-line determination of a wide variety of

analytes in a range of application fields. Enzyme-

based biosensors either using optical or electrochem-

ical transducers are at the heart of the development

of compact, self-contained devices for diversified

chemical or biological analysis, which play an

increasingly important role in our modern society.

Their importance will certainly increase in the years

ahead.

References

[1] Clark L C Jr (1956) Trans Am Soc Artif Intern Organs 2:

41–57

[2] Clark L C Jr, Lyons C (1962) Ann N Y Acad Sci 102: 29–45

[3] Rechnitz G A, Kobos R K, Riechel S J, Gebauer C R (1977)

Anal Chim Acta 94: 357–365

[4] Turner A P F (2000) Science 290: 1315–1317

[5] Thevenot D R, Toth K, Durst R A, Wilson G S (2001) Biosens

Bioelectron 16: 121–131

[6] G€oopel W (1995) Biosens Bioelectron 10: 853–883

[7] Schmidt H-L, Schuhmann W (1996) Biosens Bioelectron 11:

127–135

[8] Zhong C-J, Porter M D (1995) Anal Chem 67: 709A–715A

[9] Bourdillon C, Demaille C, Gueris J, Moirous J, Sav�eeant J-M

(1993) J Am Chem Soc 115: 12264–12269

[10] Bourdillon C, Demaille C, Moirous J, Sav�eeant J-M (1994)

J Am Chem Soc 116: 10328–10329

[11] Bourdillon C, Demaille C, Moirous J, Sav�eeant J-M (1996)

Acc Chem Res 29: 529–535

[12] Yang M, McGovern M E, Thompson M (1997) Anal Chim

Acta 346: 259–275

[13] Bardea A, Katz E, B€uuckmann A F, Willner I (1997) J Am

Chem Soc 119: 9114–9119

[14] Pantano P, Morton T H, Kuhr W G (1991) J Am Chem Soc

113: 1832–1833

[15] Hoshi T, Takeshita H, Anzai J, Osa T (1995) Anal Sci 11:

311–312


[16] Du X, Anzai J, Osa T, Motohashi R (1996) Electroanalysis 8:

813–816

[17] Chen Q, Kobayashi Y, Takeshita H, Hoshi T, Anzai J (1998)

Electroanalysis 10: 94–97

[18] Anzai J, Takeshita H, Kobayashi Y, Osa T (1998) Anal Chem

70: 811–817

[19] Willner I, Rubin S, Cohen Y (1993) J Am Chem Soc 115:

4937–4938

[20] Katz E, Heleg-Shabtai V, Willner B, Willner I, B€uuckmann A

F (1997) Bioelectrochem Bioenerg 42: 95–104

[21] Willner I, Katz E, Willner B, Blonder R, Heleg-Shabtai V,

B€uuckmann A F (1997) Biosens Bioelectron 12: 337–356

[22] Krull U J, Heimlich M S, Kallury K M R, Piunno P A E,

Brennan J D, Brown R S, Nikolelis D P (1995) Can J Chem

73: 1239–1250

[23] Curulli A, Carelli I, Trischitta O, Palleschi G (1997) Biosens


[24] Scheller F W, Schubert F, Neumann B, Pfeiffer D, Hintsche

R, Dransfeld I, Wollenberger U, Renneberg R, Warsinke A,

Johansson G, Skoog M, Yang X, Bogdanovskaya V,

B€uuckmann A, Zaitsev S Y (1991) Biosens Bioelectron 6:

245–253

[25] Dicks J M, Aston W J, Davis G, Turner A P F (1986) Anal

Chim Acta 182: 103–112

[26] Gorton L, Karan H I, Hall P D, Inagaki T, Okamoto Y,

Skotheim T A (1990) Anal Chim Acta 228: 23–30

[27] Wring S A, Hart J P (1992) Analyst 117: 1215–1229

[28] Rosen-Margalit I, Rishpon J (1993) Biosens Bioelectron 8:

315–323

[29] Glezer V, Lev O (1993) J Am Chem Soc 115: 2533–2534

[30] Avnir D, Braun S, Lev O, Ottolenghi M (1994) Chem Mater

6: 1605–1614

[31] Swalen J D, Allara D L, Andrade J D, Chandross E A, Caroff

S, Israelshvili J, McCarthy T J, Murray R, Pease R F, Rabolt

J F, Wynne K J, Yu H (1987) Langmuir 3: 932–950

[32] Willner I, Kata E (2000) Angew Chem Int Ed 39: 1180–1218

[33] Free A H (1977) Ann Clin Lab Sci 7: 479–485

[34] Kuswandi B, Andres R, Narayanaswamy R (2001) Analyst

126: 1469–1491

[35] Wolfbeis O S, Oehme I, Papkovskaya N, Klimant I (2000)

Biosens Bioelectron 15: 69–76

[36] Lin A W C, Yeh P, Yacynych A M, Kuwana T (1977)

J Electroanal Chem 84: 411–419

[37] Yacynych A M, Kuwana T (1978) Anal Chem 50:

640–645

[38] Ianniello R M, Wieck H J, Yacynych A M (1983) Anal Chem

55: 2067–2070

[39] Kamin R A, Wilson G S (1980) Anal Chem 52: 1198–1285

[40] Koval C A, Anson F C (1978) Anal Chem 50: 223–229

[41] Rocklin R D, Murray R W (1979) J Electroanal Chem 100:

271–282

[42] Bourdillon C, Bourgeois J P, Thomas D (1979) Biotechnol

Bioeng 21: 1877–1879

[43] Bourdillon C, Bourgeois J P Thomas D (1980) J Am Chem

Soc 102: 4231–4235

[44] Kuznetsov B A, Byzova N A, Shumakovich G P (1994)

J Electroanal Chem 371: 85–92

[45] Ianniello R M, Yacynych A M (1981) Anal Chem 53:

2090–2095

[46] Ianniello R M, Yacynych A M (1981) Anal Chim Acta 131:

123–132

[47] Ianniello R M, Lindsay T J, Yacynych A M (1982) Anal

Chem 54: 1098–1101

[48] Ianniello R M, Yacynych A M (1983) Anal Chim Acta 146:

249–253

[49] Razumas V J, Jasaitis J J, Kulys J J (1983) Bioelectrochem

Bioenerg 10: 427–439

[50] Jonsson G, Gorton L (1985) Biosensors 1: 355–368

[51] Osborn J A, Yacynych A M, Roberts D C (1986) Anal Chim

Acta 183: 287–292

[52] Antrim R F, Yacynych A M, Wieck H J, Lutter G W (1988)

Analyst 113: 341–344

[53] Katz E, Riklin A, Willner I (1993) J Electroanal Chem 354:

129–144

[54] Willner I, Riklin A, Shoham B, Rivenzon D, Katz E (1993)

Adv Mater 5: 912–915

[55] Mann-Buxbaum E, Pittner F, Schalkhammer T, Jachimowicz

A, Jobst G, Olcaytug F, Urban G (1990) Sens Actuators B 1:

518–522

[56] Williams R A, Blanch H W (1994) Biosens Bioelectron 9:

159–167

[57] Miyasaka T, Koyama K, Watanabe T (1990) Chem Lett,

pp 627–630

[58] Solov’ev A A, Katz E, Shuvalov V A, Erokhin Y E (1991)

Bioelectrochem Bioenerg 26: 29–41

[59] Katz E (1994) J Electroanal Chem 365: 157–164

[60] Hong H-G, Bohn P W, Sligar S G (1993) Anal Chem 65:

1635–1638

[61] Hong H-G, Jiang M, Sligar S G, Bohn P W (1994) Langmuir

10: 153–158

[62] Firestone M A, Shank M L, Sligar S G, Bohn P W (1996)

J Am Chem Soc 118: 9033–9041

[63] Wolfbeis O S, Leiner M J P, Posch H E (1986) Mikrochim

Acta III: 359–366

[64] Bacon J R, Demas J N (1987) Anal Chem 59: 2780–2785

[65] Lippitsch M E, Pusterhofer J, Leiner M J P, Wolfbeis O S

(1988) Anal Chim Acta 205: 1–6

[66] Carraway E R, Demas J N, DeGraff B A, Bacon J R (1991)

Anal Chem 63: 337–342

[67] Klimant I, Belser P, Wolfbeis O S (1994) Talanta 41: 985–991

[68] Freeman T M, Seitz W R (1978) Anal Chem 50: 1242–1246

[69] Wolfbeis O S (1991) Fiber optic chemical sensors and

biosensors, vol. 1. CRC Press, Boca Raton, FL, USA

[70] Owen V M (1985) Ann Clim Biochem 22: 559–564

[71] Hall E A (1986) Enzyme Microb Technol 8: 651–658

[72] Scheller F W, Hintsche R, Pfeiffer D, Schubert F, Riedel K,

Kindervater R (1991) Sens Actuators B 4: 197–200

[73] Wagner G, Guilbault G G (1994) Food biosensor analysis,

Marcel Dekker, New York, USA

[74] Turner A P F (1994) Curr Opin Biotech 5: 49–53

[75] Mascini M (1995) Sens Actuators B 29: 121–125

[76] Aboul-Enein H Y, Stefan R-I, van Staden J F (1999) Crit Rev

Anal Chem 29: 323–331

[77] Wang J (1999) Anal Chem 71: 328R–332R

[78] Wolfbeis O S (2000) Anal Chem 72: 81R–90R

[79] Wilson G S, Hu Y (2000) Chem Rev 100: 2693–2704

[80] D’Orazio P (2003) Clin Chim Acta 334: 41–69

[81] Wolfbeis O S (2004) Anal Chem 76: 3269–3284

[82] Schaffar B P H, Dremel B A A, Schmid R D (1989) GBF

Monographs 13: 229–232

[83] Li X, Fortuney A, Guilbault G G, Suleiman A A (1996) Anal

Lett 29: 171–180

[84] Li X, Rosenzweig Z (1997) Anal Chim Acta 353: 263–273

[85] Vidal M M, Delgadillo I, Gil M H, Alonso-Chamarro J

(1996) Biosens Bioelectron 11: 347–354

[86] Wu X J, Choi M M F (2003) Anal Chem 75: 4019–4027

[87] Yan Y, Xu Y, Li S (2002) Shengwu Yixue Gongchengxue

Zazhi 19: 101–102

[88] Carazuela M D, Cuesta B, Moreno-Bondi M C, Quejido A

(1997) Biosens Bioelectron 12: 233–240

130 M. M. F. Choi

[89] Trettnak W, Wolfbeis O S (1989) Anal Biochem 184: 124–127

[90] Marquette C A, Leca B D, Blum L J (2001) Luminescence

16: 159–165

[91] Zhang Z, Ma W (1993) Gaodeng Xuexiao Huaxue Xuebao

14: 1366–1369

[92] Wang H, Cao X, Jia K, Chai X, Lu H, Lu Z (2001) Proc SPIE-

Int Soc Opt Eng 4601: 115–118

[93] Marazuela M D, Moreno-Bondi M C (1998) Anal Chim Acta

374: 19–29

[94] Tsafack V C, Marquette C A, Leca B, Blum L J (2000)

Analyst 125: 151–155

[95] Hasunuma T, Kuwabata S, Fukusaki E-I, Kobayashi A

(2004) Anal Chem 76: 1500–1506

[96] Wolfbeis O S, Posch H E (1988) Fresen Z Anal Chem 332:

255–257

[97] Lau R C W, Choi M M F, Lu J (1999) Talanta 48: 321–331

[98] Walters B S, Nielsen T J, Arnold M A (1988) Talanta 35:

151–155

[99] Gautier S M, Blum L J, Coulet P R (1990) J Bioluminesc

Chemiluminesc 5: 57–63

[100] Xie X, Suleiman A A, Guilbault G G, Yang Z, Sun Z (1992)

Anal Chim Acta 266: 325–329

[101] Marshall R W, Gibson T D (1992) Anal Chim Acta 266:

309–315

[102] Verostko C E, Atwater J E, Akse J R, Dehart J L, Wheeler R R

(1998) US Pat 5792621

[103] Schier G M, Moses R G, Gan I E T, Blair S C (1988) Diabetes

Res Clin Pract 4: 177–181

[104] Clarke W, Becker D J, Cox D, Santiago J V, White N H,

Betschart J, Eckenrode K, Levandoski L A, Prusinki E A,

Simineiro L M, Snyder A L, Tideman A M, Yaegar T (1988)

Diabetes Res Clin Pract 4: 209–213

[105] Meadows D, Schultz J S (1988) Talanta 35: 145–150

[106] Trettnak W, Wolfbeis O S (1989) Anal Chim Acta 221:

195–203

[107] Tolosa L, Malak H, Rao G, Lakowicz J R (1997) Sens

Actuators B 45: 93–99

[108] Tolosa L, Gryczynski I, Eichorn L R, Dattelbaum J D,

Castellano F N, Rao G, Lakowicz J R (1999) Anal Biochem

267: 114–120

[109] D’Auria S, Dicesare N, Gryczynski Z, Gryczynski I, Rossi

M, Lakowicz J R (2000) Biochem Biophys Res Commun

274: 727–731

[110] Aslan K, Lakowicz J R, Geddes C D (2004) Anal Chim Acta

517: 139–144

[111] Robinson M R, Eaton R P, Haaland D M, Koepp G W,

Thomas E V, Stallard B R, Robinson P L (1992) Clin Chem

38: 1618–1622

[112] Heise H M, Marbach R, Koschinsky T H, Gries F A (1994)

Ann Occup Hyg 18: 439–447

[113] March W F, Rabinovitch B, Adams R, Wise J R, Melton M

(1982) Trans Am Soc Artif Intern Organs 28: 232–235

[114] Rabinovitch B, March W F, Adams R L (1982) Diabetes Care

5: 254–265

[115] Moscone D, Pasini M (1992) Talanta 39: 1039–1044

[116] Tatsu Y, Yamashita K, Yamaguchi M, Yamamura S,

Yamamoto H, Yoshikawa S (1992) Chem Lett, pp 1615–1618

[117] Narang U, Prasad P N, Bright F V (1994) Anal Chem 66:

3139–3144

[118] Male K B, Gartu P O, Kamen A A, Luong J H T (1997)

Biotechnol Bioeng 55: 497–504

[119] Wang B, Li B, Deng Q, Dong S (1998) Anal Chem 70:

3170–3174

[120] Chen T, Friedman K A, Lei I, Heller A (2000) Anal Chem 72:

3757–3763

[121] Sapre A G, Bedekar A, Deshpande A V, Lali A M (2000)

Biotechnol Lett 22: 569–573

[122] Chen X, Matsumoto N, Hu Y, Wilson G S (2002) Anal Chem

74: 368–372

[123] Karyakin A A, Kotel’nikova E A, Lukachova L V, Karyakina

E E, Wang J (2002) Anal Chem 74: 1597–1603

[124] Matsumoto R, Mochizuki M, Kano K, Ikeda T (2002) Anal

Chem 74: 3297–3303

[125] Wei X, Cruz J, Gorski W (2002) Anal Chem 74: 5039–5046

[126] Palmisano F, Zambonin P G, Centonze D, Quinto M (2002)

Anal Chem 74: 5913–5918

[127] Gouda M D, Kumar M A, Thakur M S, Karanth N G (2002)


[128] Haouz A, Stieg S (2002) Enzyme Microb Technol 30:

129–133

[129] Zen J-M, Kumar A S, Chung C-R (2003) Anal Chem 75:

2703–2709

[130] Hrapovic S, Luong J H T (2003) Anal Chem 75: 3308–3315

[131] Uwira N, Opitz N, Lubbers D W (1984) Adv Exp Med Biol

169: 915–921

[132] Neubauer A, Pum D, Sleytr U B, Klimant I, Wolfbeis O S

(1996) Anal Chim Acta 11: 317–325

[133] Dremel B A A, Kalisz H, Schaffar B P H, Draxler S,

Lippitsch M E, Schmid R D, Wolfbeis O S (1992) In

GBF monographs: biosensors, fundamentals, technologies

and applications, vol. 17. In: Scheller F, Schmid R D (eds)

VHC, Weinheim, Germany, p 259–262

[134] Dremel B A A, Schaffar B P H, Schmid R D (1989) Anal

Chim Acta 225: 293–301

[135] Moreno-Bondi M C, Wolfbeis O S, Leiner M J P, Schaffar

B P H (1990) Anal Chem 62: 2377–2380

[136] Schaffar B P H, Wolfbeis O S (1990) Biosens Bioelectron 6:

137–148

[137] Trettnak W, Leiner M J P, Wolfbeis O S (1988) Analyst 113:

1519–1523

[138] Chen Q, Kenausis G L, Heller A (1998) J Am Chem Soc 120:

4582–4585

[139] Wu X, Choi M M F, Xiao D (2000) Analyst 125: 157–162

[140] Wu X J, Choi M M F (2004) Anal Chim Acta 514:

219–226

[141] Choi M M F, Pang W S H, Xiao D, Wu X (2001) Analyst 126:

1558–1563

[142] Rosenzweig Z, Kopelman R (1996) Anal Chem 68: 1408–1413

[143] Xu H, Aylott J W, Kopelman R (2002) Analyst 127: 1471–1477

[144] Papkovsky D B, O’Riordan T C, Guilbault G G (1999) Anal

Chem 71: 1568–1573

[145] Papkovsky D B, Ovchinnikov A N, Ogurtsov V I, Ponomarev

G V, Korpela T (1998) Sens Actuators B 51: 137–145

[146] Trettnak W, Wolfbeis O S (1989) Anal Chim Acta 221:

195–203

[147] Hartnett A M, Ingersoll C M, Baker G A, Bright F V (1999)

Anal Chem 71: 1215–1224

[148] Trettnak W, Leiner M J P, Wolfbeis O S (1988) Biosensors 4:

15–26

[149] Li C-I, Lin Y-H, Shih C-L, Tsaur J-P, Chau L-K (2002)


[150] Wang C H, Chen S M, Wang C M (2002) Analyst 127:

1507–1511

[151] Wolfbeis O S, Sch€aaferling M, D€uurkop A (2003) Microchim

Acta 143: 221–227

[152] Rickus J L, Tobin A J, Zink J I, Dunn B (2002) Materials

Research Society Symposium Proceedings 723: 155–160

[153] Wang A J, Arnold M A (1992) Anal Chem 64: 1051–1055

[154] Cordek J, Wang X, Tan W (1999) Anal Chem 71: 1529–1533

[155] Kar S, Arnold M A (1992) Anal Chem 64: 2438–2443


[156] Dremel B A A, Schmid R D, Wolfbeis O S (1991) Anal Chim

Acta 248: 351–359

[157] Cattaneo M V, Male K B, Luong J H T (1992) Biosens


[158] Cattaneo M V, Luong J H T (1993) Biotechnol Bioeng 41:

659–665

[159] Blankenstein G, Preuschoff F, Spohn U, Mohr K-H, Kula

M-R (1993) Anal Chim Acta 271: 231–237

[160] Trettnak W, Wolfbeis O S (1989) Anal Lett 22: 2191–2197

[161] Dremel B A A, Yang W, Schmid R D (1990) Anal Chim Acta

234: 107–112

[162] Ignatov S G, Ferguson J A, Walt D R (2001) Biosens


[163] Papkovsky D B, O’Riordan T, Soini A (2000) Biochem Soc T

28: 74–77

[164] Trettnak W, Wolfbeis O S (1989) Fresen Z Anal Chem 334:

427–430

[165] Schubert F, Wang F, Rinneberg H (1995) Mikrochim Acta

121: 237–247

[166] Arnold M A (1988) Proc SPIE-Int Soc Opt Eng 906: 128–133

[167] Wangsa J, Arnold M A (1988) Anal Chem 60: 1080–1082

[168] Liu X, Tan W (1999) Mikrochim Acta 131: 129–135

[169] Michel P E, Gautier S M, Blum L J (1996) Anal Lett 29:

1139–1155

[170] Marquette C A, Blum L J (1999) Anal Chim Acta 381: 1–10

[171] Marquette C A, Degiuli A, Blum L J (2000) Appl Biochem

Biotech 89: 107–115

[172] Koncki R, Leszczy�nnska E, Cybulska A, Glab S (1996) Anal

Chim Acta 321: 27–34

[173] Mueller C, Hitzmann B, Schubert F, Scheper T (1995) Proc

SPIE-Int Soc Opt Eng 2388: 558–567

[174] Mueller C, Hitzmann B, Schubert F, Scheper T (1997) Sens

Actuators B 340: 71–77

[175] Polster J, Prestel G, Wollenweber M, Kraus G, Gauglitz G

(1995) Talanta 42: 2065–2072

[176] Healey B G, Walt D R (1995) Anal Chem 67: 4471–4476

[177] Healey B G, Li L, Walt D R (1997) Biosens Bioelectron 12:

521–529

[178] Xie X, Suleiman A A, Guilbault G G (1992) Biotechnol

Bioeng 39: 1147–1150

[179] Carlyon E E, Lowe C R, Reid D, Bennion I (1992) Biosens


[180] Goldfinch M J, Lowe C R (1984) Anal Biochem 138: 430–436

[181] Sansubrino A, Mascini M (1994) Biosens Bioelectron 9:

207–216

[182] Xie X, Suleiman A A, Guilbault G G (1990) Anal Lett 23:

2143–2153

[183] Krysteva M, Al Hallak M (2003) The Scientific World 3:

585–592

[184] Koncki R, Lenarczuk T, Radomska A, Glab S (2001) Analyst

126: 1080–1085

[185] Radomska A, Glab S, Koncki R (2001) Analyst 126:

1564–1567

[186] Kovacs B, Nagy G, Dombi R, Toth K (2003) Biosens


[187] Wolfbeis O S, Li H (1993) Biosens Bioelectron 8: 161–166

[188] Chen H, Wang E (2000) Anal Lett 33: 997–1011

[189] De Marcos S, Hortiguela R, Galban J, Castillo J R, Wolfbeis

O S (1999) Microchim Acta 130: 267–272

[190] Brennan J D, Brown R S, Della Manna A, Kallury K M R,

Piunno P A, Krull U J (1993) Sens Actuators B 11:

109–119

[191] Rhines T D, Arnold M A (1989) Anal Chim Acta 227:

387–396

[192] Xie X, Suleiman A A, Guilbault G G (1991) Talanta 38:

1197–1200

[193] Gauglitz G, Reichert M (1992) Sens Actuators B 6: 83–86

[194] Popa E, Kubota Y, Tryk D A, Fujishima A (2000) Anal Chem

72: 1724–1727

[195] Gong Z, Zhang Z (1996) Anal Lett 29: 695–709

[196] Zhang Z, Ma W, Yang M (1992) Fenxi Huaxue 20: 1048–1051

[197] Ge X, Tolosa L, Rao G (2004) Anal Chem 76: 1403–1410

132 Progress in Enzyme-Based Biosensors Using Optical Transducers

progress in enzyme-based biosensors using optical transducers

Documents