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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.
http://en.wikipedia.org/wiki/Volatile_organic_compoundshttp://en.wikipedia.org/wiki/Volatile_organic_compoundshttp://en.wikipedia.org/wiki/Wastewaterhttp://en.wikipedia.org/wiki/Pharmaceuticalhttp://en.wikipedia.org/wiki/Painthttp://en.wikipedia.org/wiki/Sulfurhttp://en.wikipedia.org/wiki/Hydrogen_sulfidehttp://en.wikipedia.org/wiki/Hydrogen_sulfidehttp://en.wikipedia.org/wiki/Sulfurhttp://en.wikipedia.org/wiki/Painthttp://en.wikipedia.org/wiki/Pharmaceuticalhttp://en.wikipedia.org/wiki/Wastewaterhttp://en.wikipedia.org/wiki/Volatile_organic_compoundshttp://en.wikipedia.org/wiki/Volatile_organic_compounds -
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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
http://en.wikipedia.org/wiki/Drinking_waterhttp://en.wikipedia.org/wiki/Turbidityhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Slow_sand_filterhttp://en.wikipedia.org/wiki/Carbon_filteringhttp://en.wikipedia.org/wiki/Carbon_filteringhttp://en.wikipedia.org/wiki/Water-borne_diseasehttp://en.wikipedia.org/wiki/Dissolved_organic_carbonhttp://en.wikipedia.org/wiki/Ammoniahttp://en.wikipedia.org/wiki/Nitratehttp://en.wikipedia.org/wiki/Excretionhttp://en.wikipedia.org/wiki/Gillhttp://en.wikipedia.org/wiki/Aquatic_animalhttp://en.wikipedia.org/wiki/Decompositionhttp://en.wikipedia.org/wiki/Decompositionhttp://en.wikipedia.org/wiki/Aquatic_animalhttp://en.wikipedia.org/wiki/Gillhttp://en.wikipedia.org/wiki/Excretionhttp://en.wikipedia.org/wiki/Nitratehttp://en.wikipedia.org/wiki/Ammoniahttp://en.wikipedia.org/wiki/Dissolved_organic_carbonhttp://en.wikipedia.org/wiki/Water-borne_diseasehttp://en.wikipedia.org/wiki/Carbon_filteringhttp://en.wikipedia.org/wiki/Carbon_filteringhttp://en.wikipedia.org/wiki/Slow_sand_filterhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Turbidityhttp://en.wikipedia.org/wiki/Drinking_water -
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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
http://en.wikipedia.org/wiki/Lime_%28material%29http://en.wikipedia.org/wiki/Membrane_%28selective_barrier%29http://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Integral_membrane_proteinhttp://en.wikipedia.org/wiki/Tissue_%28biology%29http://en.wikipedia.org/wiki/Mucous_membranehttp://en.wikipedia.org/wiki/Basement_membranehttp://en.wikipedia.org/wiki/Cell_compartmenthttp://en.wikipedia.org/wiki/Chemistryhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Environment_%28biophysical%29http://en.wikipedia.org/wiki/Peroxisomehttp://en.wikipedia.org/wiki/Peroxidehttp://en.wikipedia.org/wiki/Organellehttp://en.wikipedia.org/wiki/Organellehttp://en.wikipedia.org/wiki/Peroxidehttp://en.wikipedia.org/wiki/Peroxisomehttp://en.wikipedia.org/wiki/Environment_%28biophysical%29http://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Chemistryhttp://en.wikipedia.org/wiki/Cell_compartmenthttp://en.wikipedia.org/wiki/Basement_membranehttp://en.wikipedia.org/wiki/Mucous_membranehttp://en.wikipedia.org/wiki/Tissue_%28biology%29http://en.wikipedia.org/wiki/Integral_membrane_proteinhttp://en.wikipedia.org/wiki/Lipid_bilayerhttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Membrane_%28selective_barrier%29http://en.wikipedia.org/wiki/Lime_%28material%29 -
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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:
http://en.wikipedia.org/wiki/Vacuolehttp://en.wikipedia.org/wiki/Granule_%28cell_biology%29http://en.wikipedia.org/wiki/Vesicle_%28biology%29http://en.wikipedia.org/wiki/Phagosomehttp://en.wikipedia.org/wiki/Autophagosomehttp://en.wikipedia.org/wiki/Clathrin-coated_vesicleshttp://en.wikipedia.org/wiki/Secretory_vesicleshttp://en.wikipedia.org/wiki/Synaptosomehttp://en.wikipedia.org/wiki/Acrosomehttp://en.wikipedia.org/wiki/Melanosomeshttp://en.wikipedia.org/wiki/Chromaffin_granulehttp://en.wikipedia.org/wiki/Chromaffin_granulehttp://en.wikipedia.org/wiki/Melanosomeshttp://en.wikipedia.org/wiki/Acrosomehttp://en.wikipedia.org/wiki/Synaptosomehttp://en.wikipedia.org/wiki/Secretory_vesicleshttp://en.wikipedia.org/wiki/Clathrin-coated_vesicleshttp://en.wikipedia.org/wiki/Autophagosomehttp://en.wikipedia.org/wiki/Phagosomehttp://en.wikipedia.org/wiki/Vesicle_%28biology%29http://en.wikipedia.org/wiki/Granule_%28cell_biology%29http://en.wikipedia.org/wiki/Vacuole -
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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.
http://en.wikipedia.org/wiki/Machinehttp://en.wikipedia.org/wiki/Concentrationhttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Diabetes_mellitushttp://en.wikipedia.org/wiki/Diabetes_mellitushttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/Blood_glucose_monitoringhttp://en.wikipedia.org/wiki/Blood_glucose_monitoringhttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Diabetes_mellitushttp://en.wikipedia.org/wiki/Diabetes_mellitushttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Concentrationhttp://en.wikipedia.org/wiki/Machine -
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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
http://en.wikipedia.org/wiki/Fluoresceinhttp://en.wikipedia.org/wiki/Fluorophorehttp://en.wikipedia.org/wiki/File:Fluorescein_spectra.jpghttp://en.wikipedia.org/wiki/File:Fluorescein_spectra.jpghttp://en.wikipedia.org/wiki/File:Fluorescein_spectra.jpghttp://en.wikipedia.org/wiki/File:Fluorescein_spectra.jpghttp://en.wikipedia.org/wiki/Fluorophorehttp://en.wikipedia.org/wiki/Fluorescein -
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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