Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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microbes, and their resistance to dehydration and

ultraviolet (UV) radiation from the sun. High relative

humidity leads to a higher number of airborne mi-

crobes. Airborne mold and yeast particles in general

have a medium equivalent diameter of approximately

2–20 mm. Therefore, gravitational and inertial forces

play a major role in their distribution, resulting in

higher fungal counts close to surfaces than at higher

levels in the air.

0002 The formation of biofilm in air-conditioning sys-

tems does not occur without a water reservoir. Nor-

mally, there is no water in air-conditioning systems,

but it can accumulate through condensation. A study

carried out in various industrial ventilation ducts has

shown that bacterial and fungal biofilms can accumu-

late rapidly within the ducts, especially where oil has

been left on the duct surfaces. The microbial popula-

tion in the air channels is dependent on the environ-

ment, filtration membranes, and positioning of air.

The membranes in the air-conditioning system and

the walls in the air-conditioning channels are places

where biofilms start to grow.

0003 Biofilm formation causes problems in many areas,

such as industrial water systems, medicine, and in the

food-processing industry. According to the literature,

biofilm problems in food processes have been found

in air-handling systems, cooling systems, milk trans-

fer lines, on conveyors, in packaging machines, on

vegetable-processing surfaces, heat exchanger sur-

faces, ultrafiltration and reverse osmosis membranes,

in blancher extractors, on mixers, slicers, gaskets,

floors, and in drains. In addition to causing problems

in cleaning and hygiene, biofilm can cause energy

losses and blockages in condenser tubes, cooling fill

materials, water circuits, and heat exchangers. Bio-

films may enter a food-processing system by causing

reduced effectiveness in ion-exchange and membrane

processes. In food-processing water supply systems,

biofilms cause problems in granular activated-carbon

columns, reverse osmosis membranes, ion exchange

systems, degasifiers, water storage tanks, and micro-

porous membrane filters. It can be seen from this list

that problems generating from biofilm can occur any-

where in the food process if design and maintenance

are not carried out properly. Biofilm formation in

these systems is a symptom of disturbance in the

process. Therefore, equipment design plays the most

important role in combating biofilm formations.

Dead ends, corners, cracks, crevices, gaskets, valves,

and joints are vulnerable points for biofilm accumu-

lation. Poorly designed sampling valves can destroy

an entire process or give rise to incorrect information

due to biofilm effects at measuring points.

0004 Biofilm can generally be produced by any microbes

under suitable conditions, although some microbes

naturally have a higher tendency to produce biofilm

than others. Common contaminants on food contact

surfaces include bacilli, enterobacteria, lactic acid bac-

teria, micrococci, staphylococci, thermophilic strepto-

cocci, and pseudomonads. The bacterial slime of one

Bacillus strain enhanced the heat resistance of the

bacterium, extending the autoclaving time required

for successful sterilization to several hours. It is some-

what alarming that pathogens such as Listeria mono-

cytogenes, Salmonella typhimurium, and Yersinia

enterocolitica can readily produce biofilms, causing

severe disinfection and cleaning problems on surfaces

in the food industry. The most common microbes in

cooling waters are the slime-forming Gallionella and

Pseudomonas, as well as common algae. From a

hygienic and health point of view, infection and dis-

ease problems with Legionella pneumophila biofilms

can occur in hot-water systems. The slime-forming

microbial flora involved in biofilm formation in

manufacturing of packaging material can be divided

into primary slime-formers, e.g., bacteria of the Bacil-

lus, Pseudomonas, and Enterobacter strains and fungi

of the Aspergillus, Mucor, and Penicillium strains, and

secondary slime-formers, e.g., bacteria of the Alcali-

genes, Flavobacterium, Klebsiella, Micrococcus,and

Staphylococcus strains and fungi of the Paecilomyces,

Tricoderma, and Trichosporum strains.

0005The lubricants used in conveyors are a problem,

especially in dairies and breweries. Acinetobacter

spp., Algaligenes spp., Pseudomonas spp. and

sulfate-reducing bacteria have been isolated from

lubricants. The biofilm microbes in the lubricant

can indirectly promote corrosion. Listeria monocyto-

genes, an opportunistic pathogen, has been isolated

from lubricants in dairies. Yeasts belonging to

Saccharomyces, Candida, and Rhodotorula have

been isolated from biofilms on conveyor tracks.

0006There are very few published studies concerning

yeast biofilms in food processing. As mentioned

above, there are many publications dealing with

food-processing biofilms; the biofilms dealt with are

mainly bacterial in nature. Yeasts belonging to Can-

dida, Rhodotorula, and Saccharomyces have also

been isolated from biofilms on can and bottle

warmers in packaging departments of the beverage

industry. Additionally, these biofilms contained bac-

teria and molds. Yeasts have also been observed in

biofilms in draught beer dispensing lines at pubs,

where they often grow mixed with bacteria. Con-

taminants introduced into the dispensing system are

attracted to the pipe surface by electrostatic inter-

actions but cannot actually adhere due to close-

range repulsion. Yeasts overcome this charge barrier

by extending surface fimbriae, which anchor them to

the conditioning film (Figure 1).

BIOFILMS 485

0007 The biofilm formation tendency of Dekkera anom-

ala, Issatchenkia orientalis (anam. Candida krusei

var. krusei), Pichia anomala, P. membranaefaciens,

Rhodotorula mucilaginosa, and Saccharomyces

cerevisiae isolated from brewery samples has been

studied. The biofilm production was found to be

strain-dependent rather than species-dependent and

additionally dependent on medium and incubation

time. The area covered by biofilm varied from 2 to

4% in 10 days for slower-growing strains to approxi-

mately 100% in 2 days for fast biofilm producers.

Reliable identified strains of the same yeast species

differed markedly in their tendency to form biofilm

under constant conditions. Sugars included in the

media increased biofilm formation. Later, mixed bio-

films containing lactic acid bacteria, enterobacteria,

and yeasts have been used in cleanability.

0008 The choice of materials and their surface treat-

ments, e.g., grinding and polishing, are important

factors in inhibiting the formation of biofilm and in

promoting the cleanability of surfaces. The surface

structure of stainless steel is very important in

avoiding biofilm formation; it has been reported

that, although the grain boundaries of AISI 316L

stainless steel constitute 3–20% of the total surface

area, over 90% of the adherent bacteria were found

attached to the grain boundaries. Hoses, tubes, and

filters containing polyvinylchloride increase the risk

of contamination. The number of free planktonic

cells in the water does not necessarily correlate with

the amount of biofilm on the pipe surfaces. Costerton

et al. found that the numbers of microbial cells ad-

hered to surfaces were 500–50 000 times the numbers

of planktonic cells in water. Inadequate cleaning and

sanitation of surfaces coated with biofilm cause con-

tamination, because the biofilm protects the microbes

against sanitizers and disinfectants.

Elimination of Biofilms

0009Physical, chemical, and microbiological cleanliness

are essential in food plants. Cleaning agents are ap-

plied to remove soil, microbes, and biofilms from

surfaces. Factors governing the selection of detergents

and disinfectants in the food industry are that the

agent should be efficient, safe, not damage or corrode

equipment, be easily rinsable, and not affect the sens-

ory values of the product produced. Physical cleanli-

ness means that there is no visible waste, foreign

matter, or slime on the equipment surfaces. Chem-

ically clean surfaces are surfaces from which undesir-

able chemical residues have been removed, whereas

microbiologically clean surfaces imply freedom from

spoilage microbes and pathogens. Removal of biofilm

is important for the maintenance of the equipment,

since the debris left on surfaces can act as nutrients for

the build-up of new biofilm. Attached bacteria or

bacteria in biofilms can be a problem in food process-

ing, because they adhere to the surfaces, and if

cleaning is insufficient the remaining bacteria begin

to grow and multiply after cleaning and contaminate

the product. To minimize biofouling, it is best to clean

the equipment at frequent and regular intervals using

suitable, efficient cleaning procedures for the process

before the biofilm has the opportunity to develop.

0010Metal chelators have been used in the break-up of

biofilm layers. Disinfection is required in food plant

operations where wet surfaces provide favorable con-

ditions for the growth of microbes. Disinfectants

approved for use in the food industry contain chlor-

ine-based compounds, iodine compounds, alcohols,

quaternary ammonium compounds, oxidants, or sur-

factants. The use of disinfectants in food plants is

dependent on the material used and the adhering

microbes. Disinfective agents are not very effective

at penetrating the polysaccharide and glycoprotein

matrix left on the surfaces after an ineffective

cleaning procedure, and thus do not destroy all the

microbes within biofilms.

0011Microbes have also been found in disinfectant so-

lutions. This means that microbial contaminants can

be spread on the surface to be cleaned instead of being

cleaned. It was reported that chlorhexidine mixtures

were contaminated with Pseudomonas spp. Pseudo-

monas spp. have also been found in concentrated

iodine solutions. Viable Serratia marcescens cells

were found in a disinfectant containing 2% chlorhex-

idine after 27 months. Microbial contamination has

1microns

fig0001 Figure 1 Scanning electron micrograph of a mixed biofilm on a

polyvinyl chloride (PVC) surface. a, yeast cells; b, bacteria, F,

protruding fimbriae. Reproduced from Storga

rds E (2000) Process

Hygiene Controls in Beer Production and Dispensing. VTT publica-

tions 410. Espoo: Otamedia Oy, with permission.

486 BIOFILMS

also been found in solutions of aldehydes, quaternary

compounds, and amfotensides. Other microbes that

have been isolated from disinfectants include Alcali-

genes faecalis, Enterobacter cloacae, Escherichia coli,

Flavobacterium meningosepticum,andPantoea ag-

glomerans. Pseudomonas spp. are relatively resistant

to chlorine treatments and can even multiply when

chlorine has been used. Even increased amounts of

chlorine (2.0 mg l

1

) did not kill Escherichia coli

grown in biofilm. Capsular Klebsiella pneumoniae

grown on glass surfaces has been shown to have a

150-fold resistance to chlorine compared with that

grown in suspensions. On metal and carbon surfaces

the corresponding resistance ratios were about 2400

and 3000 times the value in suspension, respectively.

Detection of Biofilm

0012 Many reviews have been published on the formation

and detection of biofilms in the laboratory. Methods

of studying biofilm formation include microbio-

logical, physical, chemical, and microscopic methods.

The biofilm consists of about 85–96% water, which

means that only 2–5% of the total biofilm volume is

detectable on dry surfaces. If the actual monitoring

practice involves only sampling of the liquid phase, it

does not reflect the location or extent of microbes

growing on surfaces. Advances in molecular biology

are producing methods by which detection and

enumeration of specific organisms on surfaces can

be performed.

0013 Hygiene testing in the food industry is currently

based on conventional cultivation using swabbing or

contact plates. These classical evaluation methods

suffer from several serious deficiencies. Conventional

cultivation measures only the number of living cells

able to grow on the chosen agar. The quantification of

cells in the biofilm is difficult to perform because they

adhere strongly to the surfaces (Figure 2). In the

cultivation of biofilm microbes it is important that

the sample is detached and mixed properly. It has

been reported that ultrasonics provide a suitable

tool for sampling of biofouled surfaces. Cultivation

showed that the use of ultrasonics detached about 10

times the number of cells from the surface compared

with swabbing.

0014Despite extensive efforts to develop reliable control

methods for practical hygiene assessment in the food

industry, there are still no such methods available.

The conventional swabbing procedure of equipment

surfaces should be modified, e.g., by chemical

loosening of the remaining biofilm cells from the

surface at the moment of assessment. A study per-

formed on industrial premises showed that a a mild

detergent solution improved microbial sampling. The

surface-wetting protocol was developed in laboratory

studies using stainless-steel surfaces covered with bio-

film. The evaluated test kit comprises a composition

designed for the pretreatment of process and equip-

ment surfaces before sampling.

0015The chemical methods used in the assessment of

biofilm formation are indirect methods based on

the utilization or production of specific compounds,

e.g., organic carbon, oxygen, polysaccharides,

and proteins, or on microbial activity (adenosine 5

triphosphate or ATP) content. ATP measurement is

a luminescence method based on the luciferine-

luciferase reaction. The ATP content of the biofilm

The stainless-steel surface after swabbing with:

(a) Cotton swab (b) Dacron swab (c) Calcium alginate swab

The coverage of bacterial biofilm and bacterial cells on the surface was:

91% (biofilm) and 6% (cells) 97% (biofilm) and 10% (cells)

95% (biofilm) and 19% (cells)

Cotton swab removed

5.6

0.3 3 10

CFU cm

−2

6.1 0.1 3 10

CFU cm

−2

6.0 0.1 3 10

CFU cm

−2

Dacron swab removed Ca-alginate swab removed

fig0002 Figure 2 (see color plate 10) Epifluorescence images of 5-day-old Pseudomonas fragi biofilm on stainless-steel surfaces (AISI 304,

2B) after sampling with cotton, Dacron, and calcium alginate swabs.

BIOFILMS 487

is proportional to the number of living cells in the

biofilm and provides information on their metabolic

activity. Kinetic data obtained for freely suspended

cells should not be used to assess immobilized bio-

mass growth, e.g., biofilm. The ATP method is in-

sensitive and therefore not suitable for hygiene

measurements in equipment where absolute sterility

is needed. Impedance measurement is used in the food

industry to control product quality and to assess the

effect of cleaning agents and disinfectants.

0016 Microscopic techniques such as epifluorescence

microscopy and, especially, scanning (SEM) and

transmission electron microscopy (TEM) are very in-

formative tools in biofilm research and hygiene stud-

ies. Electron microscopy has been used to identify

biofilm structures. SEM provides accurate informa-

tion on surface materials and the position of biofilm

cells, but it does not provide quantitative data for

statistical analysis.

0017 One advantage of epifluorescence image analysis is

that it measures the adherent cells on the surface,

rather than cells that have been detached by some

method from the surface. Epifluorescence microscopy

of multilayered biofilms can only be counted two-

dimensionally. This technique has been used to deter-

mine the effects of antimicrobial agents and biofilm

formation by monitoring attachment rates, detach-

ment rates, growth, motility, viability, morphology,

and cell area. More accurate information is obtained

on the chemical and biological relationships between

microbes and their microenvironment in situ in real

time using confocal microscopy. Laser microscopy

permits optical sectioning of biological materials

without optical interference from other focal planes.

Confocal microscopy has been used to study both

growth and metabolism of living cells in biofilms.

Conclusions

0018 Equipment surfaces in the food industry provide the

microbes growing in biofilms with the interface

needed for growth and with easily obtainable liquids

and nutrients needed for growth. Biofilm formation

on food-processing surfaces should be combated,

using efficient cleaning procedures to improve pro-

cess hygiene. Cleaning in the food industry should be

based on systematic planning. The knowledge that

microbes grow differently on surfaces and in suspen-

sions is the first step towards developing advanced

regimes in process hygiene. It is very important to

bear in mind that the biofilm contains 85–96%

water. Biofilms are less likely to accumulate in well-

designed systems that can be cleaned effectively. De-

tection of deposits built up on equipment surfaces at

an early stage enables effective countermeasures and

thus results in improvement of process hygiene.

Methods used for monitoring process hygiene are

often based on conventional cultivation, using vari-

ous types of agar plates or ATP. Conventional culti-

vation requires several days before the result can be

obtained, and it measures those cells that are able

to form colonies on the given agar. ATP is used

for measuring total hygiene. Many valuable meth-

odological tools used in biofilm research can be ap-

plied in industrial monitoring. Online monitoring

techniques transferred from various industrial fields,

e.g., glass-fiberoptics, infrared techniques, ion mobil-

ity techniques, bioluminescence, microelectrodes,

and heat transfer could be used for control of micro-

bial and chemical contaminants on process surfaces.

See also: Bacillus: Occurrence; Cleaning Procedures in

the Factory: Types of Detergent; Types of Disinfectant;

Overall Approach; Escherichia coli: Occurrence;

Listeria: Properties and Occurrence; Microscopy:

Scanning Electron Microscopy; Transmission Electron

Microscopy; Salmonella: Properties and Occurrence;

Yeasts; Yersinia enterocolitica: Properties and

Occurrence

Further Reading

Austin JW and Bergeron G (1995) Development of bacterial

biofilms in dairy processing lines. Journal of Dairy

Research 62: 509–519.

Block JC (1992) Biofilms in drinking water distribution

systems. In: Melo LF, Bott TR, Fletcher M and Capde-

ville B (eds) Biofilms – Science and Technology, pp.

469–485. Dordrecht: Kluwer Academic.

Carpentier B and Cerf O (1993) A review: biofilms and

their consequences, with particular reference to hygiene

in the food industry. Journal of Applied Bacteriology 75:

499–511.

Characklis WG (1981) Fouling biofilm development: a pro-

cess analysis. Biotechnology and Bioengineering 23:

1923–1960.

Chisti Y and Moo-Young M (1994) Cleaning-in-place

systems for industrial bioreactors: design, validation

and operation. Journal of Industrial Microbiology 13:

201–207.

Costerton JW, Cheng K-J, Geesey GG et al. (1987) Bacterial

biofilms in nature and disease. Annual Review of Micro-

biology 41: 435–464.

European Hygienic Equipment Design Group (EHEDG)

(1993) Hygienic equipment design criteria. Trends in

Food Science Technology 4: 225–229.

Gibson H, Taylor JH, Hall KE and Holah JT (1995) Bio-

films and their Detection in the Food Industry. Chipping

Campden: CCFRA.

Gilbert P, Brading M, Walker J and Wimpenny J (eds)

(1999) Biofilms: The Good, the Bad and the Ugly.

Cardiff: BioLine.

Hugenholz P, Cunningham MA, Hendrikz JK and Fuerst JA

(1995) Desiccation resistance of bacteria isolated from

488 BIOFILMS

an air-handling system biofilm determined using a

simple quantitative membrane filter method. Letters in

Applied Microbiology 21: 41–46.

Lappalainen J, Loikkanen S, Havana M et al. (2000) Mi-

crobial testing methods for detection of residual cleaning

agents and disinfectants – prevention of ATP biolumin-

escence measurement errors in the food industry. Journal

of Food Protein 63: 210–215.

Mattila-Sandholm T and Wirtanen G (1992) Biofilm

formation in the industry, a review. Food Review

International 8: 573–603.

Mettler E and Carpentier B (1998) Varations over time of

microbial load and physicochemical properties of floor

materials after cleaning in food industry premises.

Journal of Food Protein 61: 57–65.

Salo S, Laine A, Alanko T, Sjo

berg A-M and Wirtanen G

(2000) Validation of the microbiological methods Hygi-

cult dipslide, contact plate and swabbing in surface

hygiene control: a Nordic collaborative study. Journal

of AOAC International 83: 1357–1365.

Scholte RPM (1996) Spoilage fungi in the industrial pro-

cessing of food. In: Samson RA, Hoekstra ES, Frisvad JC

and Filtenborg O (eds) Introduction to Foodborne

Fungi, 5th edn, pp. 275–288. Baarn: Centraalbureau

voor Schimmelcultures.

Storga

rds E (ed.) (2000) Process Hygiene Control in Beer

Production and Dispensing, VTT publication 410.

Espoo: Otamedia Oy.

Tarvainen K, Wirtanen G and Mattila-Sandholm T (1994)

Preventing airborne microbial risks in rooms with

special hygiene requirements. In: Healthy Buildings

’94, pp. 217–222. Budapest: Technical University of

Budapest.

Wirtanen G (1995) Biofilm formation and its elimination

from food processing equipment. VTT publication 251.

Espoo: Technical Research Centre of Finland.

Wirtanen G, Salo S and Mikkola A (eds) (1999) 30th

-Nordic Contamination Control Symposium, pp.

1–198. VTT Symposium 193, Espoo: Libella Painopal-

velu Oy.

Wirtanen G, Saarela M and Mattila-Sandholm T (2000)

Biofilms – impact on hygiene in food industries. In:

Bryers J (ed.) Biofilms II: Process Analysis and Applica-

tions, pp. 327–372. New York: John Wiley-Liss.

Biogenic Amines See Amines

BIOSENSORS

I E Tothill and A P F Turner, Cranfield University,

Silsoe, UK

Introduction

0001 Consumers and legislators are placing farmers, food

manufacturers, and retailers under increasing pres-

sure to provide food that is high in quality, free from

polluting compounds and chemical residues, and

consistent in nutritional value. The demand for fresh

natural foods, ready-prepared, and cook-chill foods

containing less preservatives and additives, more nu-

tritional value and free from pathogenic microorgan-

isms and genetically modified (GM) material has

fueled demands for rapid sensing methods. Food an-

alysis is usually carried out by ‘off-site’ methods, in

which the samples are sent to a laboratory for testing.

These methods suffer from the disadvantage of being

expensive and time-consuming, and require the use of

highly trained personnel. However, central analytical

facilities do generally provide the highest accuracy in

quantification and the lowest detection limits. Many

assays are available on the market today that promise

results within 24 h or less, such as immunoassays,

polymerase chain reaction assays (PCR), and adeno-

sine triphosphate (ATP) detection methods. These

assays are used to measure the concentration of a

variety of hormones, allergens, viruses, bacteria, and

chemical pollutants. However, for today’s environ-

ment and lifestyle, there is an increasing need for

more easy-to-use, robust, rapid, and relatively inex-

pensive technologies. The availability of these assays

would facilitate ‘on-site’ sample analysis, and the

number of samples requiring further analysis using

laboratory-based instruments could then be reduced.

Hence, there is a clear need for simpler, alternative

analysis systems and preferably methods that allow

real-time monitoring in the field such as biosensors.

0002Clark and Lyons pioneered the underlying concept

of the biosensor in a paper published in 1962. They

reported an ‘enzyme electrode,’ comprising an immo-

bilized enzyme at an electrochemical detector, and

BIOSENSORS 489

hence produced a sensor that was responsive to

the substrate of the enzyme. Since then, the field of

biosensors has greatly expanded due to advances in

signal transduction technologies and also to huge

advances in the receptor technology used as the

sensing element. There is now a wide array of bio-

logical and synthetic sensing receptors and signal

transducers available to construct a particular biosen-

sor. The approach generally employed is to identify

the analytical target and then to choose the sensing

molecule that gives the required affinity, selectivity,

and dynamic range for the analyte or analytes of

interest. The assay format signal transduction strat-

egy can then be selected based on the analytical

requirements of the final device (Table 1).

0003 The study of biosensors has been strongly driven by

the needs of medical diagnostics. Instant analysis of

medical samples can provide better patient care and

disease management. There has also, however, been a

wide appreciation of biosensor technology in other

areas such as veterinary care, animal husbandry, food

and process control, and environmental analysis.

While work initially concentrated on biosensors for

the aqueous phase, more challenging environments

such as the gas phase and organic solvents have been

explored recently. The diagnostics industry is very

diverse, with numerous markets requiring different

products. The main controlling influences are price

of both the instrumentation and the test, the accuracy,

sensitivity and number of parameters required, speed,

and portability. The types of instruments required for

the diagnostics market can be divided into large mul-

tianalyte analyzers, bench-top portable instruments

and one-shot disposable sensors. Many of the instru-

mentations developed to date have been designed for

the medical diagnostics market. However, a number

of these have been adapted for the agrofood market.

In addition, the market for diagnostics continues to

grow. To date, an astounding diversity of research on

biosensors has been reported in the literature, and

these papers offer an almost infinite number of

suggestions for biosensor construction.

Definition

0004Biosensors are analytical devices incorporating a bio-

logical material, a biologically derived material or a

biomimic as the recognition molecules, which is

either intimately associated with or integrated within

a physicochemical transducer or transducing micro-

systems. The usual aim is to produce a digital elec-

tronic signal that is proportional to the concentration

of a specific analyte or group of analytes (Figure 1).

This signal can result from a change of proton con-

centration, uptake or release of gases such as oxygen

and ammonia, light emission, reflectance or absorp-

tion, heat emission, or other mechanisms, brought

about by the action of the sensing molecules. This

signal can then be converted by the transducer into

a measurable response. Both the biological and the

electrical signal can be manipulated further by ampli-

fication or processing. While the signal may in

principle be continuous, devices can be configured

to yield single measurements to meet specific market

requirements.

0005The analytical capabilities of biosensors have been

further increased by miniaturization and the im-

proved processing power of modern microelectronics.

Different biosensor formats have been developed for

single target analytes and for broad-spectrum moni-

toring. From this definition, it can be seen that a

number of different biosensor devices either already

exist or are theoretically possible.

Sensing Elements Used in Biosensors

(Receptors)

Natural Receptors

0006There is a wide range of naturally produced molecules

from plants, animals, and microorganisms that can be

used as the receptor in biosensors. These molecules

may be broadly classified into two groups: the bioca-

talytic group (e.g., enzymes, whole cells, mitochon-

dria, and tissue slices); and the affinity reaction group

(e.g., nucleic acids, antibodies, and cell receptors). In

each case, the biosensor exploits the selective binding

capabilities inherent in biomolecules. Table 2 list the

most frequently used sensing molecules and their re-

spective analytes. In catalytic sensors, the change in

the concentration of a component resulting from the

catalyzed reaction is detected to give the sensor

signal. In the case of an affinity sensor, the binding

event itself (between the receptor and the target ana-

lyte) is monitored.

0007Because of their specificity and catalytic properties,

enzymes have found widespread use as sensing

elements in biosensors (enzyme biosensors). Since

tbl0001 Table 1 Components used for biosensor construction

Receptors (recognition elements) Transducers

Tissues Optical

Microorganisms Electrochemical

Organelles Thermometric

Cell receptors Piezoelectric

Enzymes Magnetic

Antibodies

Nucleic acids

Synthetic receptors

490 BIOSENSORS

the development of the first glucose sensor based

on the immobilization of glucose oxidase on an

oxygen-sensing electrode, there has been a prolifer-

ation of applications involving a wide variety of sub-

strates. A variety of enzymes belonging to classes of

oxidoreductases, hydrolases, and lyases have been

integrated with different transducers to construct bio-

sensors for applications in health care, veterinary

medicines, the food industry, environmental monitor-

ing, and defence. However, the most important group

has been the oxidoreductases, catalyzing oxidation or

reduction events using either oxygen or cofactors.

Enzymes are either used alone, as in catalytic biosen-

sors, or used in conjunction with other components,

such as antibodies as the signal marker.

0008 Whole cells of living organisms, such as bacteria,

yeast, fungi, plant and animal cells, or even tissue

slices, have been used as the recognition component

in biosensors (whole-cell sensors). The sensing

principle is usually based on interrogating the general

metabolic status of the cells by detecting oxygen or

substrate consumption, the production of carbon

dioxide or metabolites, bacterial luminescence, or

direct electrochemical sampling of the electron trans-

port chain. A range of whole-cell or microbial sensors

have been constructed to detect toxic and pollutant

compounds. Interest in these types of devices for

environmental and toxicity monitoring is increasing

because of their ability to monitor directly biological

effects, as opposed to simply providing a total

chemical analysis, which may not be related to the

bioavailability of the contaminant.

0009Antibody–antigen binding reactions are used

mainly in affinity sensors. However, other biological

components such as cell receptors, single-stranded

DNA (to bind and detect its complementary se-

quence), and lectins (plant proteins used to bind

carbohydrates) are also being used. Combinations of

the above biological components have been utilized

to provide new or improved analytical capabilities.

The developments of polyclonal antibodies (Pabs)

and the huge advances in monoclonal antibodies

(Mabs) and recombinant antibody production have

created a major force in the diagnostics market. Anti-

bodies are widely applied in a range of devices from

rapid wet chemistry assays such as immunoassay kits,

to dipsticks and biosensors. The use of antibody frag-

ments and molecularly engineered antibodies is a

future area of growth for immunosensors. A new

research approach is to recover the genes of useful

antibodies, express them in bacteria or plants, de-

velop three-dimensional structural models, and then

derive improved variants by directed and combina-

torial mutagenesis. The affinity and selectivity of the

recombinant antibody can thus be adapted to make it

more suitable as an analytical tool.

0010The main disadvantage of using biological mol-

ecules in sensor devices is their poor stability, which

is often the stumbling block preventing wider com-

mercialization. Research in improving the stability of

these compounds is expanding, and methods include

the use of soluble, positively charged polymers, such

as diethyl amino ethyl, dextran, lacitol, and sugar

derivatives. A new approach to overcome the stability

problem of biological molecules is to replace them

with artificial receptors or biomimics.

Artificial Receptors (Biomimics)

0011New advances in receptor discovery such as the de-

velopment of artificial receptors using the combined

approach of computer (molecular) modeling and

molecularly imprinted polymers or combinatorial

synthesis has increased the range of receptors that

can be used for the construction of suitable sensing

layers for biosensors.

0012Molecular modeling is a powerful tool to study

molecular recognition, the specific interaction be-

tween substrate and receptor. The affinity interactions

between synthetic receptors and target analytes

comprise hydrogen bond interactions, p-stacking

interactions, Van der Waals interactions and electro-

static interactions, which constitute molecular forces

Bioreceptor

(affinity or

catalytic)

Transducer ElectronicsAnalytes

signal

fig0001 Figure 1 The biosensor.

tbl0002 Table 2 Biological systems most frequently used in biosensor

construction for sensing their respective binding substance

Sensingmolecule Analyte to be detected

Enzyme Substrate analog, inhibitor, cofactor,

metal chelaters, dyes

Antibody Antigen, virus, cell

Nucleic acid Complementary base sequence,

histone, nucleic acid polymerase,

binding protein

Cell Cell-surface-specific protein, lectin

Receptor, carrier protein Hormone, vitamin

BIOSENSORS 491

involved in molecular recognition processes. To

design the artificial receptors, computer modeling is

used to provide structure information for the target

analyte, which will then be used to guide the design of

combinatorial libraries and rational design of the

artificial receptors. Molecular modeling allows the

prediction of ligands that are expected to bind

strongly to key regions of biologically important mol-

ecules (e.g., enzymes, macromolecular receptors) of

known three-dimensional structure, so as to inhibit or

alter their activity.

0013 Molecular-imprinting polymerization is currently

the focus of intense research interest and is being

used in a wide range of application areas, e.g., in the

preparation of selective separation materials, artifi-

cial antibodies, and synthetic enzymes and in molecu-

lar recognition. Molecular imprinting relies upon the

presence of complementary interactions (noncovalent

or reversible covalent) between sites in the template

molecules (the analyte) and the functional mono-

mer(s) used in the polymerization process (Figure 2).

The functional monomer(s) is a key component in the

imprinting process, though generally constituting a

relatively small percentage of the resultant MIP.

The complexation of a template by these building

blocks prior to, and during, polymerization to yield

the complementary positioning of functionalities,

is the central dogma of molecular imprinting.

0014 The combinatorial library technique (combinator-

ial chemistry) is a fast developing area of receptor

discovery and has been widely used by the drug indus-

try to both discover and optimize lead compounds.

An example is the optimization of the antidepres-

sant drug, benzodiazepine. Combinatorial libraries

consist of a large array of diverse molecular entities,

generated by the systematic and repetitive covalent

connection of a set of different ‘building blocks’ of

varying structures. The number of possible com-

pounds (n) generated in a single library can be found

using the relationship:

n ¼ b

where b represents the number of building blocks for

each step and x, the number of synthetic steps.

0015Enormous effort has been devoted to the develop-

ment of new strategies for peptide and nonpeptide

libraries. The principle is illustrated in Figure 3.A

large number of compounds can be generated in a

very short time using combinatorial library tech-

niques, which would be near impossible using clas-

sical organic synthesis methodologies. Molecular

modeling is also used as a combined approach with

combinatorial chemistry to facilitate a more efficient

receptor discovery process. Combinatorial chemistry

has been used to generate affinity ligands, which can

be used as synthetic receptors in biosensor devices.

0016Research activity in the area of artificial receptor

discovery has increased in the last few years, in par-

ticular the production of synthetic receptors for med-

ical and environmental diagnostics. The development

of artificial receptors for various purposes remains an

important challenge. Combinatorial synthesis and

molecularly imprinted polymers are two of the most

exciting and rapidly growing areas in ligand discov-

ery, which can overcome the stability problems inher-

ent in natural ligands. However, the challenge is

to produce receptors that compete with the natural

molecules with respect to sensitivity and stability.

Transducers

0017The transduction element of a biosensor must be

capable of converting a specific biological response

Polymerization

Washing

fig0002 Figure 2 Molecularly imprinted polymers (MIPs).

fig0003Figure 3 Combinatorial chemistry.

492 BIOSENSORS

into a quantifying signal. The transducers must also

be suitable for receptor immobilization at, or close to,

its surface. A list of transducers that have been used in

the construction of biosensors is given in Table 3.

Considerable work is being carried out to improve

transducer designs, with the blending of a variety

of different technologies. The operation principle of

commonly used transducers is described briefly

below.

Electrochemical Transducers

0018 Electrochemical devices usually monitor the current

at a fixed voltage (amperometry) or the voltage at

zero current (potentiometry), or measure conductiv-

ity or impedance changes. Amperometric biosensors

were the first type to be developed and have been in

use as glucose biosensors for over 35 years. These

devices have continued to be the most popular, largely

due to their simplicity, ease of production, and the

low cost of the devices and instruments. The signal in

amprometric devices is dependent on the rate of mass

transfer to the electrode surface. The most commonly

used devices monitor the analyte concentration by

measuring the fall in O

tension, the production of

or determination of the redox reaction of a

mediator molecule (electron shuttle). A large number

of mediators are available, but the most widely used

include potassium ferricyanide, tetrathiafulvalene

(TTF), tetracyanoquinodimethane (TCNQ), and the

ferrocenes.

0019 A potentiometric sensor operates under conditions

of near-zero current flow and measures the difference

in potential between the working electrode and a

reference electrode. Ion-selective electrodes (ISEs),

of which the pH electrode is a well-known example,

are the most important of this class of transducer.

Specificity is conferred by selective membranes,

which may be formed from metal salts or polymer

membranes containing ion-exchangers or neutral car-

riers. To date, there is a range of commercially avail-

able ISEs that can detect specific ions (i.e., calcium,

nitrate, potassium, copper, barium, chloride, etc.).

Devices can also include the gas-sensing electrodes

(GSEs), which are ISEs modified by the use of a gas-

permeable membrane, and the chemically sensitive

field-effect transistors (FETs), which are semicon-

ductor devices that respond to the surface electric

gradient or charge at the gate electrode. More

recently, light-addressable potentiometric sensors

(LAPSs) have been developed, based on silicon tech-

nology. A chemical reaction at the surface of the

silicon chip will shift the surface potential and hence

affects the current, which allows the rate of the

reaction to be monitored.

0020Conductimetric biosensors are based on measuring

the time dependence of the change in conductivity as

a result of the receptor recognition of its complemen-

tary analyte. Impedance is the total electrical resist-

ance to the flow of an alternating current being

passed through a given medium. Typically, during

measurement, impedance decreases, while conductiv-

ity and capacitance increase.

Optical Transducers

0021Optical transducers use a number of principles, such

as the effect of the biological event on light absorp-

tion, fluorescence, refractive index, or other optical

parameters. These types of transducers have become

increasingly popular over the last few years with

many devices now commercially available. The

early-developed optoelectronic devices utilize a color

change induced in pH-sensitive dyes by the action of

an enzyme that generates or consume protons. More

recent approaches to measure pH changes related

to the analyte use optical sensor devices based on

dynamic quenching by molecular oxygen of a

fluorescence of an indicator.

0022Most of the recent work on optical biosensors has

concentrated on the use of evanescent wave technol-

ogy. When light is totally internally reflected during,

for example, transmission through an optical wave-

guide, a small amount of energy propagates beyond

the optical interface. In an unclad waveguide, this can

be coupled to fluorescent labels constrained close to

the optical interface as a result, for example, of anti-

body–antigen interactions. Alternatively, a direct

immunosensor can be constructed, which does not

require the use of labels, by measuring the change of

refractive index due to the sensing receptor inter-

action with the target analyte. Surface plasmon res-

onance (SPR) is currently receiving the most interest

tbl0003 Table 3 Transducers used in biosensors

Transducer Output

Electrochemical (electrodes)

Amperometry Applied current

Potentiometry Voltage

Conductimetry/impedimetry Conductivity/impedance

Optical (optrodes)

Colorimetric Color

Luminescence Light intensity

Fluorescence Light intensity

Interferometry

Calorimetric

Thermistor

(heat-sensitive sensor)

Temperature

Mass

Piezoelectric

(surface acoustic wave devices)

Acoustic wave

Magnetic

BIOSENSORS 493

in this area and forms the basis of a successful com-

mercial instrument. The technology is based on the

excitation of the electron plasma (surface plasmon) of

a thin metal layer covering the surface of the wave-

guide. The change in angle of the incident light or the

reflectance minima due to the change in the refractive

index in the vicinity of the metalized surface can

be monitored.

Calorimetric Transducers

0023 Thermometric devices operate by measuring enthalpy

changes during the biological reaction using one of a

range of thermometers, thermopiles (array of thermo-

couples), or thermistors.

0024 The detection of heat during the enzyme catalysis

can be used in the construction of calorimetric enzyme

biosensors. Since heat is either produced or consumed

in most reactions, these can be converted to analytical

signal to monitor analyte concentrations.

Piezoelectric Transducers

0025 Sensors based on piezoelectric principles use the

change in the resonant frequency of wave propaga-

tion through a piezoelectric material. These principles

can be used to measure mass, viscosity, or density

changes at the sensor surface. These devices are

able to generate and transmit acoustic waves in a

frequency-dependent manner. Bulk wave devices

operate by transmitting a wave from one side of

the crystal to the other, while surface acoustic wave

(SAW) transmit waves along a single crystal face.

Magnetic transducers

0026 Very recently, a few papers have been published de-

scribing biosensors based on magnetic transduction.

Hall-effect transducers, for example, have been used

to detect magnetic labels in immunosensors. Practical

application of this type of devices has been limited,

but they are included here for completeness.

Fabrication of Biosensors

0027 The commercial production of devices such as the

ExacTech home blood-glucose biosensor (MediSense,

Inc., Cambridge, MA) has verified the importance of

automated manufacturing technologies where large

numbers of inexpensive, reproducible electrochem-

ical devices are required. The capability of printing

materials at high precision and speed is very desirable

for the mass production of analytical devices such

as biosensors. Techniques such as screen-printing,

ink-jet printing, air-brushing, and Cavro deposition

have been developed under microprocessor control

and adapted for biosensor fabrication, especially

thick-film biosensors (Figure 4). These deposition

techniques are rapid and flexible with regard to the

ink characteristics employed. Thin-film deposition

has also been used, which involves the application

of material through a mask, under vacuum, via evap-

oration due to heating, or by placing the substrate to

be coated between two electrodes (sputtering). This

process is able to create patterns less than 1 mm thick,

making it suitable for the construction of micro-

sensors. Thin-film sensors are generally produced by

a variety of vapor-deposition techniques, electro-

chemical methods, and, more recently, by the use of

Langmuir–Blodgett technology. Thin metallic films,

usually deposited by vapor deposition, have been

used extensively for the production of sensors utiliz-

ing surface plasmon resonance. These sensors have

been primarily directed towards affinity sensors.

0028Silicon fabrication technology has been increas-

ingly used as a means of mass production of biosen-

sors. The use of silicon microfabrication for both

electrochemical and optical sensors is expanding

and the capability of on-chip electronic signal ampli-

fication and data processing is very attractive. This is

an area of intense research activity mainly by diag-

nostics companies. Several companies are using

microelectronics technology based on silicon to pro-

duce optical chips for biosensor application.

Biosensors Based on Biocatalitic

Interactions (Biocatalytic Sensors)

0029The biosensor market has been dominated for many

years by electrochemical biocatalytic sensors. The

majority of biocatalytic sensors are based on enzyme

or whole-cell sensors. Amperometric enzyme biosen-

sors form the majority of commercial biosensors

available today. In the enzyme electrode, for example,

an enzyme specific for the substrate of interest is

immobilized on the sensor surface. The chemical con-

version of the analyte is catalyzed as it enters the

enzyme layer, and either the cosubstrate or the prod-

uct of the reaction is transduced to an electrochemical

signal, which is proportional to the concentration of

fig0004Figure 4 Screen-printed biosensor.

494 BIOSENSORS