Chemical Aspects in Biotechnology[1]

8/3/2019 Chemical Aspects in Biotechnology[1]

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BIOREACTOREnzymes bioreactors are reactors in which enzymatic

reactions are carried out. Since enzymatic reactionsrequire carefully maintained pH, temperature and

substrate level conditions, these bioreactors are equipped

with the appropriate instrumentation that allow the

operator to monitor all these conditions and ensure timely

completion of the reaction to obtain the desired product.

These are designed with the biologically active species

either immobilized to porous particles or to the surface ofmembranes or hollow fibers. The material use for enzyme

immobilization is called carrier matrix are usually inert

polymer of inorganic materials.

A bioreactor may refer to any manufactured or

engineered device or system that supports a biologically

active environment. In one case, a bioreactor is a vessel inwhich a chemical process is carried out which involves

organisms or biochemically active substances derived

from such organisms. This process can either be aerobic

or anaerobic. These bioreactors are commonly cylindrical,

ranging in size from liters to cubic meters, and are often

made of stainless steel. A bioreactor may also refer to a

device or system meant to grow cells or tissues in the

context ofcell culture. These devices are being developed

for use in tissue engineering or biochemical engineering.

On the basis ofmode of operation, a bioreactor may be
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classified as batch, fed batch or continuous (e.g. a

continuous stirred-tank reactor model). An example of a

continuous bioreactor is the chemostat.

Organisms growing in bioreactors may be suspended or

immobilized. A simple method, where cells are

immobilized, is a Petri dish with agar gel. Large scale

immobilized cell bioreactors are:

moving media, also known as Moving Bed Biofilm

Reactor (MBBR) packed bed fibrous bed membrane Bioreactor design is a relatively complex engineering

task, which is studied in the discipline ofbiochemical

engineering. Under optimum conditions, the

microorganisms or cells are able to perform theirdesired function with a 100 percent rate of success

The bioreactor's environmental conditions like gas

(i.e., air, oxygen, nitrogen, carbon dioxide) flow

rates, temperature, pH and dissolved oxygen levels,

and agitation speed/circulation rate need to be closely

monitored and controlled. Most industrial bioreactor

manufacturers use vessels, sensors and a controlsystem networked together

Fouling can harm the overall sterility and efficiencyof the bioreactor, especially the heat exchangers. To
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avoid it, the bioreactor must be easily cleaned and as

smooth as possible (therefore the round shape). A

heat exchanger is needed to maintain the bioprocess

at a constant temperature. Biological fermentation isa major source of heat, therefore in most cases

bioreactors need refrigeration. They can be

refrigerated with an external jacket or, for very large

vessels, with internal coils.

In an aerobic process, optimal oxygen transfer isperhaps the most difficult task to accomplish.

Oxygen is poorly soluble in watereven less in

fermentation brothsand is relatively scarce in air

(20.95%). Oxygen transfer is usually helped by

agitation, which is also needed to mix nutrients and

to keep the fermentation homogeneous. There are,

however, limits to the speed of agitation, due both to

high power consumption (which is proportional to thecube of the speed of the electric motor) and to the

damage to organisms caused by excessive tip speed.

In practice, bioreactors are often pressurized; this

increases the solubility of oxygen in water.

Sewage treatment

Bioreactors are also designed to treat sewage and

wastewater. In the most efficient of these systemsthere is a supply of free-flowing, chemically inert

media that acts as a receptacle for the bacteria that

breaks down the raw sewage. Examples of these

bioreactors often have separate, sequential tanks and
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a mechanical separator or cyclone to speed the

division of water and biosolids. Aerators supply

oxygen to the sewage and media further accelerating

breakdown. Submersible mixers provide agitation inanoxic bioreactors to keep the solids in suspension

and thereby ensure that the bacteria and the organic

materials "meet". In the process, the liquids

Biochemical Oxygen Demand (BOD) is reduced

sufficiently to render the contaminated water fit for

reuse. The biosolids can be collected for further

processing or dried and used as fertilizer. An

extremely simple version of a sewage bioreactor is a

septic tank whereby the sewage is left in situ, with or

without additional media to house bacteria. In this

instance, the biosludge itself is the primary host

(activated sludge) for the bacteria. Septic systems are

best suited where there is sufficient landmass and thesystem is not subject to flooding or overly saturated

ground and where time and efficiency is not of an

essence.

Immobilised enzymes

Immobilized enzymes are enzymes which may be

attached to each other, to insoluble materials, or

enclosed in a membrane or gel. This can provide

increased resistance to changes in conditions such as

pH or temperature. It also allows enzymes to be held
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in place throughout the reaction, following which

they are easily separated from the products and may

be used again. Immobilised enzymes are used in

bioreactors .These procedures are used to producemany products which used to use micro-organisms.

Advantages of immobilisation

1.It makes for easier purification of the product as theseparation of the enzymes from the products is easily

accomplished.2.It is easy to recover and recycle the enzymes. Thisleads to a more economical process.

3.The enzymes remain functional for much longer as itis a gentler process.

Uses of immobilised enzymes

The following products are derived from immobilisedenzyme action:

1.Fructose derived from glucose: Fructose is sweeterthan glucose and is used in soft drinks and other

sweet products.

2.Antibiotics: Enzymes are used to change penicillininto new, wider used, antibiotics.

3.Sewage Treatment: Instead of bacteria enzymes canbe immobilised and used.


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Immobilization of enzymes for the fabrication of

biosensors

Most of the techniques described above have been used for the immobilization of biocatalyst for biosensor

applications. The choice of the support and the technique

for the preparation of membranes has been dictated by

the low diffusional resistance of the membrane coupled

with its ability to incorporate optimal amount of enzyme

per unitxv area. In this respect, stable membranes have

been prepared by binding glucose oxidase to cheese clothin the fabrication of a glucose biosensor. Enzymes

entrapped inside the reversed micelle have also shown

promise in the fabrication of biosensors. Cross-linked

enzyme crystals (CLCs) described above provides their

own supports and so achieves enzyme concentration close

to the theoretical packing limit in excess of even highly

concentrated enzyme solutions. In view of this, CLCs are

particularly attractive in biosensor applications where the

largest possible signal per unit volume is often critical.

Sensors based on small transducer or thinner enzyme

immobilized membranes (miniature biosensors) are also

emerging. The development of molecular devices

incorporating a sophisticated and highly organized

biological information processing function is a long-term

goal of bioelectronics. For this purpose, it is necessary in

the future to develop suitable methods for micro


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immobilizing the proteins/enzymes into an organized

array/pattern, as well as designing molecular structures

capable of performing the required function. A typical

example is the micro immobilization of proteins intoorganized patterns on a silicon wafer based on a specific

binding reaction between strepatavidin and biotin

combined with photolithography techniques.Immobilized

enzymes have also been used for various other analytical

purposes. A recent development has been in obtaining a

stable dry immobilized enzyme, like acetylcholineesterase,

on polystyrene micro titration plates for mass screening

of its inhibitors in water and biological fluids.

Bio fertilizer

'Bio fertilizer' is a substance which contains living

microorganisms which, when applied to seed, plant

surfaces, or soil, colonizes the rhizosphere or the interior

of the plant and promotes growth by increasing the supply

or availability of primary nutrients to the host plant. Bio

fertilizers add nutrients through the natural processes of

Nitrogen fixation , solubilizing phosphorus, and

stimulating plant growth through the synthesis of growth

promoting substances. Bio fertilizers can be expected to

reduce the use ofchemical fertilizers and pesticides. The

microorganisms in bio fertilizers restore the soil's natural

nutrient cycle and build soil organic matter. Through the
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use of bio fertilizers, healthy plants can be grown while

enhancing the sustainability and the health of soil. Since

they play several roles, a preferred scientific term for such

beneficial bacteria is plant-growth promoting rhizo

bacteria (PGPR). Therefore, they are extremely

advantageous in enriching the soil fertility and fulfilling

the plant nutrient requirements by supplying the organic

nutrients through microorganism and their byproduct.

Hence, bio fertilizers do not contain any chemicals whichare harmful to the living soil. Bio fertilizers are Eco-

friendly organic agro-input and more cost effective than

chemical fertilizers. Bio fertilizers like Rhizobium,

Azotobacter, Azospirillum and blue green algae (BGA)

are in use since long time ago. Rhizobiuminoculant is

used for leguminous crops. Azotobacter can be used withcrops like wheat,maize, mustard, cotton, potato and other

vegetable crops. Azospirillum inoculants are

recommended mainly for sorghum, millets, maize,

sugarcane and wheat. Blue green algae belonging to

genera Nostoc, Anabaena, Tolypothrix and Aulosira fix

atmospheric nitrogen and are used as inoculants for paddycrop grown both under upland and low land conditions.

Anabaena in association with water fern Azolla

contributes nitrogen up to 60 kg/ha/season and also
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enriches soils with organic matter Other types of bacteria,

so-called phosphate solubilizing bacteria like Pantoea

agglomerans strain P5, and Pseudomonas putida strain

P13 are able to solubilize the insoluble phosphate from

organic and inorganic phosphate source. In fact, due to

immobilization of phosphate by mineral ions such as Fe,

Al and Ca or organic acids, the rate of available

phosphate (Pi) in soil is well below plant needs. In

addition, chemical Pi fertilizer are also immobilized in thesoil immediately so that less than 20 percent of added

fertilizer is absorbed by plants. Therefore, reduction in Pi

resources, on one hand, and environmental pollutions

resulted from both production and applications of

chemical Pi fertilizer, on the other hand, have already

demanded the use of new generation of phosphatefertilizers globally known as phosphate solubilizing

bacteria or phosphate biofertilizers,

As it is living thing, it can symbiotically associate with

plant root. Involved microorganisms could readily and

safely convert complex organic material in simplecompound, so that plant easily taken up. Microorganism

function is in long duration causing improvement of the

soil fertility. It maintains the natural habitat of the soil. It

increases crop yield by 20-30%. Replace chemical
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nitrogen and phosphorus by 25% in addition to

stimulating of the plant growth. Finally it can provide

protection against drought and some soil borne diseases.

Advantages of Biofertilizers

Cost effective relative to chemical fertilizer and reduces

the costs towards fertilizers use, especially regarding

nitrogen and phosphorus. It is environmentally friendly

fertilizer that not only prevents damaging the naturalsource but helps to some extend clean the nature from

precipitated chemical fertilizer.And can provide better

nourishment to plants.

Biosurfactants

Biosurfactants are surface-active substances synthesisedby living cells; they are generally non-toxic and

biodegradableInterest in microbial surfactants has been

steadily increasing in recent years due to their diversity,

environmentally friendly nature, possibility of large-scale

production, selectivity, performance under extreme

conditions and potential applications in environmental

protection. Biosurfactants enhance the emulsification of

hydrocarbons, have the potential to solubilise

hydrocarbon contaminants and increase their availability


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for microbial degradation. The use of chemicals for the

treatment of a hydrocarbon polluted site may contaminate

the environment with their by-products, whereas

biological treatment may efficiently destroy pollutants,

while being biodegradable themselves. Hence,

biosurfactant producing microorganisms may play an

important role in the accelerated bioremediation of

hydrocarbon contaminated sites. These compounds can

also be used in enhanced oil recovery and may beconsidered for other potential applications in

environmental protection. Other applications include

herbicides and pesticides formulations, detergents, health

care and cosmetics, pulp and paper, coal, textiles, ceramic

processing and food industries, uranium ore-processing

and mechanical dewatering of peat.

Several microorganisms are known to synthesise surface-

active agents, most of them are bacteria and yeasts. When

grown on hydrocarbon substrate as the carbon source,

these microorganisms synthesise a wide range of

chemicals with surface activity, such as glycolipid,phospholipid and others. These chemicals are apparently

synthesised to emulsify the hydrocarbon substrate and

facilitate its transport into the cells. In some bacterial

species such as Pseudomonas aeruginosa, biosurfactants
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are also involved in a group motility behavior called

swarming motility.

Biosurfactants and Deepwater Horizon

The use of biosurfactants as a way to remove petroleum

from contaminated sites has been questioned, and

criticized as irresponsible and environmentally unsafe.

Biosurfactants were not used by BP after the Deepwater

Horizon offshore drilling rig went down on April 20,2010, on the resulting Deepwater Horizon oil spill.

However, unprecedented amounts of Corexit, a surfactant

solution produced by Nalco (whose active ingredient is

Tween-80), were sprayed directly into the ocean at the

leak and on the sea-water's surface, the theory being that

the surfactants would isolate individual molecules of oilmaking it easier for petroleum consuming microbes to

digest the oil. However some scientists say that rather

than helping the situation the surfactants have only

managed to disperse and sink the oil below the surface

and out of sight. Naturally occurring petroleum

consuming microbes have evolved on the bottom of the

ocean where they have adapted to live in areas where oil

seeps naturally from the ocean floor.

Biofiltration
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It is a pollution control technique using living material to

capture and biologically degrade process pollutants.

Common uses include processing waste water, capturing

harmful chemicals or silt from surface runoff, and

microbiotic oxidation of contaminants in air.

Examples of biofiltration include;

Bioswales, Biostrips, Biobags, Bioscrubbers, and

Trickling filters Constructed wetlands and Natural wetlands Slow sand filters Treatment ponds Green belts Living walls Riparian zones, Riparian forests, Bosques When applied to air filtration and purification,

biofilters use microorganisms to remove air pollution.

The air flows through a packed bed and the pollutant

transfers into a thin biofilm on the surface of the

packing material. Microorganisms, including bacteria

and fungi are immobilized in the biofilm and degradethe pollutant. Trickling filters and bioscrubbers rely

on a biofilm and the bacterial action in their

recirculating waters.
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The technology finds greatest application in treatingmalodorous compounds and water-soluble volatile

organic compounds (VOCs). Industries employing

the technology include food and animal products, off-

gas from wastewater treatment facilities,

pharmaceuticals, wood products manufacturing, paint

and coatings application and manufacturing and resin

manufacturing and application, etc. Compounds

treated are typically mixed VOCs and various sulfurcompounds, including hydrogen sulfide.

One of the main challenges to optimum biofilteroperation is maintaining proper moisture throughout

the system. The air is normally humidified before it

enters the bed with a watering (spray) system,

humidification chamber, bioscrubber, or biotricklingfilter. Properly maintained, a natural, organic packing

media like peat, vegetable mulch, bark or wood chips

may last for several years but engineered, combined

natural organic and synthetic component packing

materials will generally last much longer, up to 10

years. A number of companies offer these types orproprietary packing materials and multi-year

guarantees, not usually provided with a conventional

compost or wood chip bed biofilter.
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Water treatment

For drinking water, biological water treatmentinvolves the use of naturally occurring micro-

organisms in the surface water to improve water

quality. Under optimum conditions, including

relatively low turbidity and high oxygen content, the

organisms break down material in the water and thus

improve water quality. Slow sand filters or carbon

filters are used to provide a place on which thesemicro-organisms grow. These biological treatment

systems effectively reduce water-borne diseases,

dissolved organic carbon, turbidity and colour in

surface water, improving overall water quality.

Use in aquaculture

The use of biofilters is commonly used on closedaquaculture systems, such as recirculating

aquaculture systems (RAS). Many designs are used,

with different benefits and drawbacks; however the

function is the same: reducing water exchanges by

converting ammonia to nitrate. Ammonia (NH4+ and

NH3) originates from the brachial excretion from thegills of aquatic animals and from the decomposition

of organic matter. As ammonia-N is highly toxic, this

is converted to a less toxic form of nitrite and then to
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an even less toxic form of nitrate. This "nitrification"

process requires oxygen (aerobic conditions), without

which the biofilter can crash. Furthermore, as this

nitrification cycle produces H+, the pH can decrease

which necessitates the use of buffers such as lime.

Biological Membrane

A biological membrane or biomembrane is an

enclosing or separating membrane that acts as aselective barrier, within or around a cell. It consists

of a lipid bilayer with embeddedproteins that may

constitute close to 50% of membrane content. The

cellular membranes should not be confused with

isolating tissues formed by layers of cells, such as

mucous andbasementmembranes.

Membranes in cells typically define enclosed spaces

or compartments in which cells may maintain a

chemical or biochemical environment that differs

from the outside. For example, the membrane around

peroxisomes shields the rest of the cell from

peroxides, and the cell membrane separates a cell

from its surrounding medium. Mostorganelles are
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defined by such membranes, and are called

"membrane-bound" organelles.

Probably the most important feature of abiomembrane is that it is a selectively permeable

structure. This means that the size, charge, and other

chemical properties of the atoms and molecules

attempting to cross it will determine whether they

succeed in doing so. Selective permeability is

essential for effective separation of a cell or

organelle from its surroundings. Biological

membranes also have certain mechanical or elastic

properties.

Particles that are required for cellular function but

are unable to diffuse freely across a membrane enterthrough a membrane transport protein or are taken

in by means ofendocytosis.
http://en.wikipedia.org/wiki/Selective_permeabilityhttp://en.wikipedia.org/wiki/Electric_chargehttp://en.wikipedia.org/wiki/Chemical_propertieshttp://en.wikipedia.org/wiki/Atomshttp://en.wikipedia.org/wiki/Elasticity_of_cell_membraneshttp://en.wikipedia.org/wiki/Elasticity_of_cell_membraneshttp://en.wikipedia.org/wiki/Membrane_transport_proteinhttp://en.wikipedia.org/wiki/Endocytosishttp://en.wikipedia.org/wiki/Endocytosishttp://en.wikipedia.org/wiki/Membrane_transport_proteinhttp://en.wikipedia.org/wiki/Elasticity_of_cell_membraneshttp://en.wikipedia.org/wiki/Elasticity_of_cell_membraneshttp://en.wikipedia.org/wiki/Atomshttp://en.wikipedia.org/wiki/Chemical_propertieshttp://en.wikipedia.org/wiki/Electric_chargehttp://en.wikipedia.org/wiki/Selective_permeability


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Diversity of biological membranes

Many types of specialized plasma membranes can

separate cell from external environment: apical,

basolateral, presynaptic and postsynaptic ones,

membranes offlagella, cilia, microvillus,filopodia and

lamellipodia, the sarcolemma of muscle cells, as well as

specialized myelin and dendritic spine membranes of

neurons. Plasma membranes can also form different typesof "supramembrane" structures such as caveola,

postsynaptic density, podosome, invadopodium,

desmosome, hemidesmosome, focal adhesion , andcell

junctions. These types of membranes differ in lipid and

protein composition.

Distinct types of membranes also create intracellularorganelles: endosome; smooth and rough endoplasmic

reticulum; sarcoplasmic reticulum; Golgi apparatus;

lysosome; mitochondrion (inner and outer membranes);

nucleus (inner and outer membranes); peroxisome;
http://en.wikipedia.org/wiki/Plasma_membranehttp://en.wikipedia.org/wiki/Apical_membranehttp://en.wikipedia.org/wiki/Basolateralhttp://en.wikipedia.org/wiki/Presynaptichttp://en.wikipedia.org/wiki/Postsynaptichttp://en.wikipedia.org/wiki/Flagellahttp://en.wikipedia.org/wiki/Ciliahttp://en.wikipedia.org/wiki/Microvillushttp://en.wikipedia.org/wiki/Filopodiahttp://en.wikipedia.org/wiki/Lamellipodiahttp://en.wikipedia.org/wiki/Sarcolemmahttp://en.wikipedia.org/wiki/Myelinhttp://en.wikipedia.org/wiki/Dendritic_spinehttp://en.wikipedia.org/wiki/Neuronshttp://en.wikipedia.org/wiki/Caveolahttp://en.wikipedia.org/wiki/Postsynaptic_densityhttp://en.wikipedia.org/wiki/Podosomehttp://en.wikipedia.org/wiki/Invadopodiumhttp://en.wikipedia.org/wiki/Desmosomehttp://en.wikipedia.org/wiki/Hemidesmosomehttp://en.wikipedia.org/wiki/Focal_adhesionhttp://en.wikipedia.org/wiki/Cell_junctionshttp://en.wikipedia.org/wiki/Cell_junctionshttp://en.wikipedia.org/wiki/Organellehttp://en.wikipedia.org/wiki/Endosomehttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Sarcoplasmic_reticulumhttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Lysosomehttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Peroxisomehttp://en.wikipedia.org/wiki/Peroxisomehttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Mitochondrionhttp://en.wikipedia.org/wiki/Lysosomehttp://en.wikipedia.org/wiki/Golgi_apparatushttp://en.wikipedia.org/wiki/Sarcoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endoplasmic_reticulumhttp://en.wikipedia.org/wiki/Endosomehttp://en.wikipedia.org/wiki/Organellehttp://en.wikipedia.org/wiki/Cell_junctionshttp://en.wikipedia.org/wiki/Cell_junctionshttp://en.wikipedia.org/wiki/Focal_adhesionhttp://en.wikipedia.org/wiki/Hemidesmosomehttp://en.wikipedia.org/wiki/Desmosomehttp://en.wikipedia.org/wiki/Invadopodiumhttp://en.wikipedia.org/wiki/Podosomehttp://en.wikipedia.org/wiki/Postsynaptic_densityhttp://en.wikipedia.org/wiki/Caveolahttp://en.wikipedia.org/wiki/Neuronshttp://en.wikipedia.org/wiki/Dendritic_spinehttp://en.wikipedia.org/wiki/Myelinhttp://en.wikipedia.org/wiki/Sarcolemmahttp://en.wikipedia.org/wiki/Lamellipodiahttp://en.wikipedia.org/wiki/Filopodiahttp://en.wikipedia.org/wiki/Microvillushttp://en.wikipedia.org/wiki/Ciliahttp://en.wikipedia.org/wiki/Flagellahttp://en.wikipedia.org/wiki/Postsynaptichttp://en.wikipedia.org/wiki/Presynaptichttp://en.wikipedia.org/wiki/Basolateralhttp://en.wikipedia.org/wiki/Apical_membranehttp://en.wikipedia.org/wiki/Plasma_membrane


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vacuole; cytoplasmic granules; cell vesicles (phagosome,

autophagosome, clathrin-coated vesicles , COPI-coated

and COPII-coated vesicles) and secretory vesicles

(including synaptosome, acrosomes, melanosomes, andchromaffin granules).

Different types of biological membranes have diverse

lipid and protein compositions. The content of membranes

defines their physical and biological properties. Some

components of membranes play a key role in medicine,

such as the efflux pumps that pump drugs out of a cell.

Biosensors

A biosensor is an analytical device which converts a

biological response into an electrical signal . The term

'biosensor' is often used to cover sensor devices used in

order to determine the concentration of substances andother parameters of biological interest even where they do

not utilize a biological system directly. Biosensors

represent a rapidly expanding field, at the present time,

with an estimated 60% annual growth rate; the major

impetus coming from the health-care industry (e.g. 6% of

the western world are diabetic and would benefit from the

availability of a rapid, accurate and simple biosensor forglucose) but with some pressure from other areas, such as

food quality appraisal and environmental monitoring. A

successful biosensor must possess at least some of the

following beneficial features:
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1.The biocatalyst must be highly specific for thepurpose of the analyses, be stable under normal

storage conditions and, except in the case of

colorimetric enzyme strips and dipsticks (see later),show good stability over a large number of assays

(i.e. much greater than 100).

2.The reaction should be as independent of suchphysical parameters as stirring, pH and temperature

as is manageable. This would allow the analysis of

samples with minimal pre-treatment. If the reaction

involves cofactors or coenzymes these should,

preferably, also be co-immobilised with the enzyme.

3.The response should be accurate, precise,reproducible and linear over the useful analytical

range, without dilution or concentration. It should

also be free from electrical noise.

4.If the biosensor is to be used for invasive monitoringin clinical situations, the probe must be tiny and

biocompatible, having no toxic or antigenic effects. If

it is to be used in fermenters it should be sterilisable.

This is preferably performed by autoclaving but no

biosensor enzymes can presently withstand such

drastic wet-heat treatment. In either case, the

biosensor should not be prone to fouling orproteolysis.

5.The complete biosensor should be cheap, small,portable and capable of being used by semi-skilled

operators.


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6.There should be a market for the biosensor. There isclearly little purpose developing a biosensor if other

factors (e.g. government subsidies, the continued

employment of skilled analysts, or poor customerperception) encourage the use of traditional methods

and discourage the decentralisation of laboratory

testing.

The biological response of the biosensor is determined

by the biocatalytic membrane which accomplishes the

conversion of reactant to product. Immobilisedenzymes possess a number of advantageous features

which makes them particularly applicable for use in

such systems. They may be re-used, which ensures that

the same catalytic activity is present for a series of

analyses. This is an important factor in securing

reproducible results and avoids the pitfalls associated

with the replicate pipetting of free enzyme otherwise

necessary in analytical protocols. Many enzymes are

intrinsically stabilised by the immobilisation process,

but even where this does not occur there is usually

considerable apparent stabilisation. It is normal to use

an excess of the enzyme within the immobilised sensor

which is sufficient to ensure an increase in the apparent

stabilisation of the immobilised enzyme. Even where

there is some inactivation of the immobilised enzyme

over a period of time, this inactivation is usually steady


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and predictable. Any activity decay is easily

incorporated into an analytical scheme by regularly

interpolating standards between the analyses of

unknown samples. For these reasons, many suchimmobilised enzyme systems are re-usable up to 10,000

times over a period of several months. Clearly, this

results in a considerable saving in terms of the enzymes'

cost relative to the analytical usage of free soluble

enzymes.

Schematic diagram showing the main components of a

biosensor.

The biocatalyst (a) converts the substrate to product. This

reaction is determined by the transducer (b) which

converts it to an electrical signal. The output from the

transducer is amplified (c), processed (d) and displayed(e).


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The key part of a biosensor is the transducer which makes

use of a physical change accompanying the reaction. This

may be

1.the heat output (or absorbed) by the reaction(calorimetric biosensors),

2.changes in the distribution of charges causing anelectrical potential to be produced (potentiometric

biosensors),

3.movement of electrons produced in a redox reaction(amperometric biosensors),

4.light output during the reaction or a light absorbancedifference between the reactants and products

(optical biosensors), or

5.Effects due to the mass of the reactants or products(piezo-electric biosensors).

Fluorescent glucose biosensors are devices thatmeasure the concentration of glucose in diabetic

patients by means of sensitive protein that relays the

concentration by means of fluorescence, an

alternative to amperometric sension of glucose. No

device has yet entered the medical market, but, due to

the prevalence of diabetes, it is the prime drive in the

construction of fluorescent biosensors.

Keeping glucose levels in check is crucial to

minimize the onset of the damage caused by diabetes.
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As a consequence, in conjunction with insulin

administrations, the prime requirement for diabetic

patients is to regularly monitor their blood glucose

levels. The monitoring systems currently in generaluse have the drawback of below optimal number of

readings, due to their reliance on a drop of fresh

blood. Some continuous glucose monitors are

commercially available, but suffer from the severe

drawback of a short working life of the probe. As a

result, there is an effort to create a sensor that relies

on a different mechanism, such as via external

infrared spectroscopy or via fluorescent biosensors.

Over the years, using a combination of rational

design and screening approaches, many possible

combinations of fluorescent sensor for glucose have

been studied with varying degrees of success: In most

approaches, the glucose concentration is translatedinto a change in fluorescence by using environment

sensitive (solvatochromic) dyes in a variety of

combinations, the fluorescent small molecule, protein

or quantum dot have been used in conjunction with a

glucose binding moiety either a boronic acid

functionalized fluorophore or a protein, such as

glucose oxidase, concanavalin A, glucose/galactose-binding protein, glucose dehydrogenase and

glucokinase.

Theory of fluorescence
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Absorption and emission spectra offluorescein

Fluorescence is a property present in certainmolecules, called fluorophores, in which they emit a

photon shortly after absorbing one with a higher

energy wavelength.

To be more specific, in order for an electron in the

outer orbital of a molecule to jump from a ground-

state orbital to an exited state orbital, it requires afixed amount of energy, which, in the case of

chromophores (molecules that absorb light), can be

acquired by absorbing a photon with an energy equal

or slightly higher. This state is short-lived, and the

electron returns to the ground-level orbital, losing the

energy either as heat or in the case of fluorophores by

emitting a photon, which, due to the loss of thedifference between the energy of the absorbed photon

and the excitation energy required, will have a lower

energy than the absorbed photon, or, expressed in

terms of wavelength, the emitted photon will have a
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longer wavelength. The difference between the two

wavelengths is called Stokes shift. This property can

be found in quantum dots, certain lanthanides and

certain organic molecules with delocalized electrons.

These excited molecules have an increase in dipole

momentum and in some cases can undergo internal

charge rearrangement. When they possess an electron

withdrawing group and an electron donating group at

opposite ends of the resonance structure, they have a

large shift in charge distribution across the molecule,which causes the solvent molecules to reorient to a

less energetic arrangement, called solvent relaxation.

By doing so, the energy of the exited state decreases,

and the extent of the difference in energy depends on

the polarity of the solvent surrounding the molecule.

An alternative approach is to use solvatochromic

dyes, which change their proprieties (intensity, half-

life, and excitation, and emission spectra), depending

on the polarity and charge of their environments.

Hence, they are sometimes loosely referred to as

environmentally sensitive dyes. These can be

positioned on specific residues that either change

their spatial arrangement due to a conformationalchange induced by glucose or reside in the glucose-

binding pocket whereby the displacement of the

water present by glucose decreases the polarity.
http://en.wikipedia.org/wiki/Stokes_shifthttp://en.wikipedia.org/wiki/Stokes_shifthttp://en.wikipedia.org/wiki/Quantum_dotshttp://en.wikipedia.org/wiki/Lanthanideshttp://en.wikipedia.org/wiki/Organic_moleculeshttp://en.wikipedia.org/wiki/Delocalized_electronhttp://en.wikipedia.org/wiki/Delocalized_electronhttp://en.wikipedia.org/wiki/Organic_moleculeshttp://en.wikipedia.org/wiki/Lanthanideshttp://en.wikipedia.org/wiki/Quantum_dotshttp://en.wikipedia.org/wiki/Stokes_shift


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An additional property of fluorescence that has found

a large usage is Frster resonance energy transfer

(FRET) in which the energy of the excited electron of

one fluorophore, called the donor, is passed on to anearby acceptor dye, either a dark-quencher (non-

emitting chromophore) or another fluorophore, which

has an excitation spectrum that overlaps with the

emission spectrum of the donor dye, resulting in a

reduced fluorescence. For sensing purposes, this

property is, in general, used either in combination

with a biomolecule, such as a protein, which

undergoes a conformational change upon ligand

binding, changing the distance between the two

labels on this protein, or in a competition assay, in

which the analyte has to compete with a known

concentration of a fixed labelled ligand for the

labelled binding site of protein. Therefore, the FRETbetween the binding site and the competing ligand

decreases when the analyte concentration is

increased. In general, the competing ligand in the

case of glucose is dextran, a long glucose polymer

attached to the scaffolding or to the enzyme.
http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transferhttp://en.wikipedia.org/wiki/Dextranhttp://en.wikipedia.org/wiki/Dextranhttp://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer

Documents

Chemical Aspects in Biotechnology[1]