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BIOSENSORS
BEN213
Nanobiosensors
WEEK-9
SpringSemester
Faculty: AsstProf.Dr.BetülGürünlü
Faculty of Engineering & Natural Sciences
Uskudar University
T
opics that will be covered in the course
 History of biosensor development, applications and requirements of biosensors and classification
 Principles of molecular recognition and transduction signal acquisition
Sourcesof Biological Recognition elements – enzymes/proteins, ssDNAs, antibody and Others
Design considerations for use of recognition elements in biosensors
Modeling of reactions for various biosensor applications‐ electrochemical, optical, piezoelectric, colorimetric, fluorometric
and others.
 Modification of sensorsurfaces and immobilization techniques
Covalent modification of surfaces using surface chemistry
Self Assembled Monolayers (SAM) and adsorptions
Other ways to immobilize biological macromolecules on various solid surfaces
 Detection methods and Physical Sensors
Electrodes/transducers – electrochemical (amperometric, potentiometric, and conductimetric transductions)
Other sensors ‐ for e.g., optical sensors (colorimetric/fluorimetric/luminometric sensors), Surface Plasmon Resonance(SPR)
sensors,and piezoelectric resonators.
 Fabrication of biosensors
Miniaturization‐application of nano‐materials, nanoparticles, carbon nanotubes (CNTs) and others
Biocompatibility – stability, reproducibility and repeatability of biomolecules on transducer surfaces
 Data acquisition, statistical and error analysis
Inter and Intra‐assays and Coefficient of variation (CV)
Signal to noise ratio
Normalization/optimization and signal retrieval
 Examples of commercial biosensors
NANOSENSORS: Exploring the Sanctuary of
Individual LivingCell: The combination of
nanotechnology, biology, advancedmaterials and
photonicsopensthepossibility of detecting and
manipulating atomsand moleculesusingnano-devices,
which have the potential for a wide variety of medical
usesat the cellular level. R
ecently reported information
showed the development of nano-biosensors and in situ
intracellular measurementsof singlecellsusing antibody-
based nanoprobes. The nano-scale size of thisnew
class of sensors also allows for measurements inthe
smallestof environments. Onesuchenvironment that has
evokeda great deal of interestisthat of individual
cells. Using these nanosensors,it is possible to probe
individual chemicalspeciesand molecular signaling
processesin specific locations within a cell.
Studies have shownthat insertion of a nano-biosensor
into a mammalian somatic cell not only appears to
have no effect on the cell membrane, but also does not
effect the cell's normal function. The possibilities to
monitor in vivo processeswithin living cells could
dramatically improveourunderstandingof cellular
function, thereby revolutionizing cell biology.
New‐generation nano‐engineered biosensors, enabling nanotechnologies and nanomaterials
Nanotechnology involves development of materials (and even complete systems) at the atomic,
molecular, or macromolecular levels. The dimensional range of interest is
approximately 1-500 nm.
Inthefield of biosensors,carbon nanotubes,thetypical elementof
nanotechnology, have received considerable attention because of their
inert properties
conducting behaviour and
high-surface area.
Particularly, their promotional ability for electron-transfer reactionswith enzymes
and other biomolecules has made carbon nanotubes the ideal supporting material
for heterogeneous catalysts.
A carbon nanotube (CNT) is a tubular form of graphite sheet in nano dimensions.
A single-wall carbon nanotube (SWCNT), ranging in diameter from 0.4 to >3 nm,
can be visualized as formed by the rolling of a layer of graphite, called a
graphene layer into a seamless cylinder.
Similarly, a multiwall carbon nanotube (MWCNT), ranging in diameter from 1.4
to >100nm, can be treated as a coaxial assembly of cylinders of SWCNTs
Thesizeof a quantumdot (QD)compared to that of other materials. QD
nanoparticle refers to quantum dots that have been solubilized and conjugated to
affinity molecules.GFP,Green fluorescentprotein; PE,Phycoerythrin;FITC,
Fluoresceineisothiocyanate.
Spectrum, absorption and emission
B
ri
ef
B
a
c
k
gr
o
u
n
d
Wavelength (nm)
Color
Wavelength (nm)
Color
Examples
Invisible
UV
Visible
V I B G Y O R
Invisible
IR region
UV/VIS Spectrophotometer works in this region
A
p
pl
ic
at
io
n
s
Aptamer/nanoparticle‐based lateral flow device. a) Adenosine‐
induced disassembly of nanoparticle aggregates into red‐colored
dispersed nanoparticles. Biotin is denoted as black stars (*). b) DNA
sequences and linkages in nanoparticle aggregates. Lateral flow
devices loaded with the aggregates (on the conjugation pad) and
streptavidin (on the membrane in cyan color) before use (c) and in a
negative (d) or a positive (e) test.
Exploiting properties of gold nanoparticles
Purple
(aggregated)
Red
(separated)
Binding to target
Fabrication of existing biosensors by bottom‐up approach
The channel region and gate dielectric of an ISFET has been fabricated by layer‐by‐layer self‐
assembly technique, (Liu and Cui, 2007). The advantages of this approach are technological
simplicity and the ability to produce low‐cost sensors.
Main research activities are:
1. Design, preparation and characterization of nanomaterials for sensors and
biosensors applications
2.Electrochemical sensors based on nanostructurated materials (i.e. carbon
nanotubes etc.) for various monitoring and other industrial applications.
3.Nanoparticle based electrochemical detection (bio)systems and biosensors for
DNA, protein and cell detection with interest for user‐friendly diagnostics, security
and quality control for various applications.
4.Fast and low cost sensing devices for heavy metals and other compounds with
interest for environment control and other industrial applications.
5.Lab‐on‐a‐chip systems with interest for in‐field screening of analytes and other
applications.
1. Carbon nanotubes (CNTs)combine in a unique way high
electrical conductivity, highchemicalstability and extremely high
mechanical strength.
2. Thesespecial properties of both single-wall (SW) and multi-wall
(MW) CNTshaveattracted the interestof many researchers in
the field of electrochemical sensors.
3. Here, the latest advances and future trends in producing,
modifying, characterizing and integrating CNTsinto
electrochemical sensing systems.
4. CNTs can be either used as single probes after formation in situ
or even individually attached onto a proper transducing surface
after synthesis.
5. BothSWCNTsand MWCNTscan be used to modify several
electrode surfacesineither vertically oriented ‘‘nanotube
forests’’ or even a non-oriented way.
6. They can be also used in sensors after mixing themwith a
polymer matrix to formCNTcomposites.
CARBON NANOTUBESCNTs
This nanobiosensor consists of two
microelectrodes connected by a bridge of
nanotubes with antibodies. Since electrical
signals produced by the union of a tumour
with the sensor molecule biosensor it detects
the appearance of a tumour.
Integration of carbon nanotubes
CNTs can be integrated into a variety of configurations to
perform electrochemical detections. The current formats
can be classified in groups:
individual CNT configurations
conventional electrodes that are modified with
CNTs, in both oriented or non‐oriented
configurations and,
CNTs integrated into a polymer matrix, creating a
CNT composite
Schematicof typical CNT solubilization alternatives:
(A) supramolecular wrapping with polymer; (B) CNT-Li+ conducting
polyelectrolyte; (C) with amino group of 2-aminomethyl-
18-crown-6 ether; (D) by amide bonds with glucosamine; and,
(E) by diamine-terminated oligomeric poly(ethylene glycol). For
details, see text and Table 1.
CNTs as modifiers of electrode surfaces
CNTs – both non‐oriented (random mixtures, see left)
and oriented (vertically aligned) –have been used to
modify several conventional electrod surfaces, glassy
carbon being the most reported.
Semiconductorquantum dots
The foremost application of quantum dots (QDs) as sensors is based on the Forster resonance
energy transfer effect (FRET).Owing to thiseffect, thefluorescenceemanating from QDs
changes from an ON state to an OFF state.
FR
E
Ttakes place when the electronic excitation energy of a donor fluorophore is relocated to
a neighbouring acceptor moleculewithout exchanging light between thedonor and the
acceptor.
Goldman etal. (2004), usedQDsfunctionalized with antibodies to perform multiplexed
fluoroimmunoassaysfor simultaneousdetection of varioustoxins.Thistype of sensorcouldbe
usedfor environmentalpurposesfor concurrentlyrecognizing pathogenslike cholera toxin or
ricin in water.
TheFR
ETprinciple wasalsoapplied to a maltosebiosensor.Thesensingmechanismwasthe
application of semiconductor QDs conjugated to a maltose binding protein covalently bound
to a FR
E
Tacceptor dye. In absence of maltose, the dye occupied the protein binding sites.
Energy transference from the QDs to the dyes quenched the QD fluorescence. When maltose
was present, it replaced the dye leading to recovery of the fluorescence.
BIOSENSORS
BEN213
Modification of sensor surfaces and immobilization
techniques
WEEK-11
SpringSemester
Faculty: Asst.Prof.Dr.BetulGurunlu
Faculty of Engineering & Natural Sciences
Uskudar University
Chemicalmodificationsof surfacesandtheirapplications
Chemical modification- two major reasons
1.to attach selective groups (binding sites or catalysts) to the sensor surface in order to
recognize target species in the sample.
2.to increase the selectivity of the sensor by reduction of interfacerences arising from
non-specific interactions.
Surface modification- Covalent and non-covalent strategies
1.Covalent approaches- a chemical bond is made between the surface and the
attached species - irreversible process.
2.Non-covalent approaches- weaker, non-bonding, interactions b/w the surface and
the adsorbed species are utilized - reversible
Eg., charge-charge, charge-dipole, dipole-dipole, dipole-induced dipole, and induced
dipole-induced dipole interactions (van der Waals interactions)
-these are less surface specfic than the covalent approaches - can be more readily
achieved
Non covalent forces
Covalent modification of sensor surface
The formation of a bond to some functional group on the surface.
The different surface modifications are as follows:
1. Reactive organo‐silane surfaces are widely used sensor surfaces for modifcation
Metal oxide surfaces were
silanized followed by linking
chemical groups
Eg., Organo-silanization
Cyanuric chloride activation on hydroxyl surfaces
Covalent attachment on acrylic surfaces (plastic)
Activation of carboxy surfaces by DCC
Eg., Modification of surfaces (sensor/biomolecule) through carbodiimide, avidin-biotin interactions
EDC-NHS coupling (covalent)
a.-OH surfaces are first modified with
carbonyldiimidazole (CDI) to formreactive intermediate-
formsa stable carbamatebond to anaminoterminated
biomolecule
b.-NH2 gp can be modified with glutaraldehyde which
forms imine bond (Schiff's base) with an aldehyde, leaving
theother aldehydefree for repeating this chemistry with
an amino-terminated molecule
c.Symmetric diisothiocyanates have also been used as
bifunctional linkers for attachment of amine-functionalized
surfaces to either thiol-terminated or amine-terminated
molecules
d.Heterobifunctional linker sulfosuccinimidyl-4-(N-
maleimidomethyl)-cyclohexane-1-carboxylate, which first
reacts with surface-bound -NH2 through displacement of
the NHS, subsequently, a thol-tethered molecule can be
reacted with the pendant maleimide gp. Toform surface-
conjugated molecules
e. EDC/NHS reaction - the most commonones
Carbodiimides
Aryl azide cross-linkers
SELFASSEMBLEDmonolayer (SAM)
SAM
ITO‐Indium tin oxide
ImmobilizationTechnology
Must be oriented in a specific direction ‐ immobilization technology
Five major immobilization methods used in the preparation of Chemical and bio‐
sensors.
1.Covalent binding ‐ attachment of the active component to the transducer surface
using a chemical reaction such as peptide bond formation or linkage to activated
surface groups (thiol, epoxy, amino, carboxylic, etc)
2. Entrapment ‐ physical trapping of the active component into a film or coating.
3.Cross‐linking ‐ similar to entrapment, only a polymerization agent (such as
gluteraldehyde) is used to provide additional chemical linkages between the active,
entrapped component and the film or coating.
4.Adsorption ‐ association of the active component with a film or coating through
hydrophobic, hydrophilic, and/or ionic interactions.
5.Biological binding ‐ association of an active biomolecule to a film or coating
through specific, biochemical binding
The covalent binding method is based on the binding of enzymes and water‐insoluble
carriers by covalent bonds. The functional groups that may take part in this binding are
listed below:
Amino group Carboxyl group Sulfhydryl group,
Hydroxyl group Imidazole group Phenolic group
Thiol group Threonine group Indole group
Covalent attachment to a support matrix must involve only functional groups of the enzyme that
are notessentialfor catalytic action. Higher activities resultfrom prevention of inactivation
reactions with amino acid residues of the active sites. A number of protective methods have been
devised:
Covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate.
1. A reversible, covalently linked enzyme-inhibitor complex.
2. A chemically modified soluble enzyme whose covalent linkage to the matrix is achieved by
newly incorporated residues.
3. A zymogen precursor.
Zymogen: Any of a group of compounds that are inactive precursors of enzymes and
require some change (such as the hydrolysis of a fragment that masks an active
enzyme) to become active
Hence, covalent binding can be brought about by the following:
Diazotization :
Amide bond formation :
Alkylation and Arylation:
SUPPORT--N=N--ENZYME.
SUPPORT--CO-NH--ENZYME
SUPPORT--CH2-NH-ENZYME
SUPPORT--CH2-S--ENZYME
SUPPORT--CH=N--ENZYME
SUPPORT--CNH-NH--ENZYME
SUPPORT--S-S--ENZYME
Schiff's base formation :
Amidation reaction :
Thiol-Disulfide interchange :
UGI reaction
Gamma-Irradiationinduced coupling
Carrier binding with bifunctional reagents :
SUPPORT-O(CH2)2N=CH(CH2)3 CH=N-ENZYME
Glutaraldehyde- The surfaces
(proteins) of microorganisms are
linked with the surfaces of other
microorganisms by aldehyde
groups of glutaraldehyde
Entrapmentand encapsulation
The immobilized enzyme can be classified into four types:
particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for
ease of handling and ease of application.
Particles - The particle form of immobilized enzyme on solid particles.
Membranes - Enzyme membranes can be prepared by attaching enzymes to membrane-
type carriers, or by molding into membrane form. The molding is done after the enzymes
have been enclosed within semi-permeate membranes of polymer by entrapment.
Tubes- Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. The
polymer tube isfirst treated ina seriesof chemicalreactionsand theenzymeisboundby
diazo coupling to give a tube in a final step.
Fibers - Enzymes that have been immobilized by entrapment in fibers to form enzyme
fibers.
The solid supports used for enzyme immobilization can be inorganic or organic . Some
organic supportsinclude: P
olysaccharides, Proteins, Carbon, P
olystyrenes, P
olyacrylates,
MaleicAnhydride basedCopolymers,Polypeptides,Vinyl andAllyl Polymers,and
Polyamides.
ImmobilizationthroughEntrapment
The entrapment method of immobilization is based on the localization of an enzyme
within the lattice of a polymer matrix or membrane. It is done in such a way as to retain
protein while allowing penetration of substrate. It can be classified into lattice and micro
capsule types
Cross‐linking is based on the formation of chemical bonds,
as in the covalent binding method. The immobilization is
performed by the formation of intermolecular cross‐
linkages between the enzyme molecules by means of bi or
multifunctional reagents.
The most common reagent used for cross‐linking is
glutaraldehyde. Cross‐linking reactions are carried out
under relatively severe conditions. These harsh conditions
can change the conformation of active center of the
enzyme; and so may lead to significant loss of activity.
The carrier‐binding method is the oldest immobilization technique
for enzymes. In this method, the amount of enzyme bound to the
carrier and the activity after immobilization depend on the nature
of the carrier. The following picture shows how the enzyme is
bound to the carrier:
The selection of the carrier depends on the nature of the enzyme
itself, as well as the:
Particle size
Surface area
Molar ratio of hydrophilic to hydrophobic groups
Chemical composition ‐polysaccharide derivatives such as
cellulose, dextran, agarose, and polyacrylamide gel.
Physical Adsorption Mode
Based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers.
Theprocessesavailable for physical adsorption of enzymesare: StaticProcedure
Electro-deposition
Reactor Loading Process
Mixing or Shaking Bath Loading
Adsorption tendsto be lessdisruptive to theenzymaticprotein thanchemicalmeansof
attachment because the binding is mainly by hydrogen bonds, multiple salt linkages, and Van
der Waal's forces.
Becauseof theweak bondsinvolved,desorption of theprotein resulting from changesin
temperature, pH,ionicstrengthor eventhemerepresence of substrate, is often observed.
Another disadvantage is non-specific, further adsorption of other proteins or other substances as
theimmobilized enzymeisused.Thismayalter the properties of the immobilized enzyme or, if
thesubstanceadsorbed isa substratefor theenzyme, the rate will probably decrease
depending on the surface mobility of enzyme and substrate.
Adsorption of the enzyme may be necessary to facilitate the covalent reactions described later
inthispresentation. Stabilization of enzymestemporarily adsorbed ontoa matrix hasbeen
achieved by cross-linkingtheprotein in a chemical reaction subsequent to its physical
adsorption.
Site-specific chemical modification of biomolecules
Site-specificproteinmodification
Week 9 - lecture 8.pptx
Week 9 - lecture 8.pptx
Week 9 - lecture 8.pptx

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Week 9 - lecture 8.pptx

  • 2. T opics that will be covered in the course  History of biosensor development, applications and requirements of biosensors and classification  Principles of molecular recognition and transduction signal acquisition Sourcesof Biological Recognition elements – enzymes/proteins, ssDNAs, antibody and Others Design considerations for use of recognition elements in biosensors Modeling of reactions for various biosensor applications‐ electrochemical, optical, piezoelectric, colorimetric, fluorometric and others.  Modification of sensorsurfaces and immobilization techniques Covalent modification of surfaces using surface chemistry Self Assembled Monolayers (SAM) and adsorptions Other ways to immobilize biological macromolecules on various solid surfaces  Detection methods and Physical Sensors Electrodes/transducers – electrochemical (amperometric, potentiometric, and conductimetric transductions) Other sensors ‐ for e.g., optical sensors (colorimetric/fluorimetric/luminometric sensors), Surface Plasmon Resonance(SPR) sensors,and piezoelectric resonators.  Fabrication of biosensors Miniaturization‐application of nano‐materials, nanoparticles, carbon nanotubes (CNTs) and others Biocompatibility – stability, reproducibility and repeatability of biomolecules on transducer surfaces  Data acquisition, statistical and error analysis Inter and Intra‐assays and Coefficient of variation (CV) Signal to noise ratio Normalization/optimization and signal retrieval  Examples of commercial biosensors
  • 3.
  • 4. NANOSENSORS: Exploring the Sanctuary of Individual LivingCell: The combination of nanotechnology, biology, advancedmaterials and photonicsopensthepossibility of detecting and manipulating atomsand moleculesusingnano-devices, which have the potential for a wide variety of medical usesat the cellular level. R ecently reported information showed the development of nano-biosensors and in situ intracellular measurementsof singlecellsusing antibody- based nanoprobes. The nano-scale size of thisnew class of sensors also allows for measurements inthe smallestof environments. Onesuchenvironment that has evokeda great deal of interestisthat of individual cells. Using these nanosensors,it is possible to probe individual chemicalspeciesand molecular signaling processesin specific locations within a cell. Studies have shownthat insertion of a nano-biosensor into a mammalian somatic cell not only appears to have no effect on the cell membrane, but also does not effect the cell's normal function. The possibilities to monitor in vivo processeswithin living cells could dramatically improveourunderstandingof cellular function, thereby revolutionizing cell biology. New‐generation nano‐engineered biosensors, enabling nanotechnologies and nanomaterials
  • 5. Nanotechnology involves development of materials (and even complete systems) at the atomic, molecular, or macromolecular levels. The dimensional range of interest is approximately 1-500 nm. Inthefield of biosensors,carbon nanotubes,thetypical elementof nanotechnology, have received considerable attention because of their inert properties conducting behaviour and high-surface area. Particularly, their promotional ability for electron-transfer reactionswith enzymes and other biomolecules has made carbon nanotubes the ideal supporting material for heterogeneous catalysts. A carbon nanotube (CNT) is a tubular form of graphite sheet in nano dimensions. A single-wall carbon nanotube (SWCNT), ranging in diameter from 0.4 to >3 nm, can be visualized as formed by the rolling of a layer of graphite, called a graphene layer into a seamless cylinder. Similarly, a multiwall carbon nanotube (MWCNT), ranging in diameter from 1.4 to >100nm, can be treated as a coaxial assembly of cylinders of SWCNTs
  • 6. Thesizeof a quantumdot (QD)compared to that of other materials. QD nanoparticle refers to quantum dots that have been solubilized and conjugated to affinity molecules.GFP,Green fluorescentprotein; PE,Phycoerythrin;FITC, Fluoresceineisothiocyanate.
  • 7. Spectrum, absorption and emission B ri ef B a c k gr o u n d Wavelength (nm) Color Wavelength (nm) Color Examples Invisible UV Visible V I B G Y O R Invisible IR region UV/VIS Spectrophotometer works in this region
  • 8. A p pl ic at io n s Aptamer/nanoparticle‐based lateral flow device. a) Adenosine‐ induced disassembly of nanoparticle aggregates into red‐colored dispersed nanoparticles. Biotin is denoted as black stars (*). b) DNA sequences and linkages in nanoparticle aggregates. Lateral flow devices loaded with the aggregates (on the conjugation pad) and streptavidin (on the membrane in cyan color) before use (c) and in a negative (d) or a positive (e) test. Exploiting properties of gold nanoparticles Purple (aggregated) Red (separated) Binding to target
  • 9. Fabrication of existing biosensors by bottom‐up approach The channel region and gate dielectric of an ISFET has been fabricated by layer‐by‐layer self‐ assembly technique, (Liu and Cui, 2007). The advantages of this approach are technological simplicity and the ability to produce low‐cost sensors.
  • 10. Main research activities are: 1. Design, preparation and characterization of nanomaterials for sensors and biosensors applications 2.Electrochemical sensors based on nanostructurated materials (i.e. carbon nanotubes etc.) for various monitoring and other industrial applications. 3.Nanoparticle based electrochemical detection (bio)systems and biosensors for DNA, protein and cell detection with interest for user‐friendly diagnostics, security and quality control for various applications. 4.Fast and low cost sensing devices for heavy metals and other compounds with interest for environment control and other industrial applications. 5.Lab‐on‐a‐chip systems with interest for in‐field screening of analytes and other applications.
  • 11. 1. Carbon nanotubes (CNTs)combine in a unique way high electrical conductivity, highchemicalstability and extremely high mechanical strength. 2. Thesespecial properties of both single-wall (SW) and multi-wall (MW) CNTshaveattracted the interestof many researchers in the field of electrochemical sensors. 3. Here, the latest advances and future trends in producing, modifying, characterizing and integrating CNTsinto electrochemical sensing systems. 4. CNTs can be either used as single probes after formation in situ or even individually attached onto a proper transducing surface after synthesis. 5. BothSWCNTsand MWCNTscan be used to modify several electrode surfacesineither vertically oriented ‘‘nanotube forests’’ or even a non-oriented way. 6. They can be also used in sensors after mixing themwith a polymer matrix to formCNTcomposites. CARBON NANOTUBESCNTs
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  • 13. This nanobiosensor consists of two microelectrodes connected by a bridge of nanotubes with antibodies. Since electrical signals produced by the union of a tumour with the sensor molecule biosensor it detects the appearance of a tumour.
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  • 23. Integration of carbon nanotubes CNTs can be integrated into a variety of configurations to perform electrochemical detections. The current formats can be classified in groups: individual CNT configurations conventional electrodes that are modified with CNTs, in both oriented or non‐oriented configurations and, CNTs integrated into a polymer matrix, creating a CNT composite Schematicof typical CNT solubilization alternatives: (A) supramolecular wrapping with polymer; (B) CNT-Li+ conducting polyelectrolyte; (C) with amino group of 2-aminomethyl- 18-crown-6 ether; (D) by amide bonds with glucosamine; and, (E) by diamine-terminated oligomeric poly(ethylene glycol). For details, see text and Table 1.
  • 24. CNTs as modifiers of electrode surfaces CNTs – both non‐oriented (random mixtures, see left) and oriented (vertically aligned) –have been used to modify several conventional electrod surfaces, glassy carbon being the most reported.
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  • 33. Semiconductorquantum dots The foremost application of quantum dots (QDs) as sensors is based on the Forster resonance energy transfer effect (FRET).Owing to thiseffect, thefluorescenceemanating from QDs changes from an ON state to an OFF state. FR E Ttakes place when the electronic excitation energy of a donor fluorophore is relocated to a neighbouring acceptor moleculewithout exchanging light between thedonor and the acceptor. Goldman etal. (2004), usedQDsfunctionalized with antibodies to perform multiplexed fluoroimmunoassaysfor simultaneousdetection of varioustoxins.Thistype of sensorcouldbe usedfor environmentalpurposesfor concurrentlyrecognizing pathogenslike cholera toxin or ricin in water. TheFR ETprinciple wasalsoapplied to a maltosebiosensor.Thesensingmechanismwasthe application of semiconductor QDs conjugated to a maltose binding protein covalently bound to a FR E Tacceptor dye. In absence of maltose, the dye occupied the protein binding sites. Energy transference from the QDs to the dyes quenched the QD fluorescence. When maltose was present, it replaced the dye leading to recovery of the fluorescence.
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  • 37. BIOSENSORS BEN213 Modification of sensor surfaces and immobilization techniques WEEK-11 SpringSemester Faculty: Asst.Prof.Dr.BetulGurunlu Faculty of Engineering & Natural Sciences Uskudar University
  • 38. Chemicalmodificationsof surfacesandtheirapplications Chemical modification- two major reasons 1.to attach selective groups (binding sites or catalysts) to the sensor surface in order to recognize target species in the sample. 2.to increase the selectivity of the sensor by reduction of interfacerences arising from non-specific interactions. Surface modification- Covalent and non-covalent strategies 1.Covalent approaches- a chemical bond is made between the surface and the attached species - irreversible process. 2.Non-covalent approaches- weaker, non-bonding, interactions b/w the surface and the adsorbed species are utilized - reversible Eg., charge-charge, charge-dipole, dipole-dipole, dipole-induced dipole, and induced dipole-induced dipole interactions (van der Waals interactions) -these are less surface specfic than the covalent approaches - can be more readily achieved
  • 40. Covalent modification of sensor surface The formation of a bond to some functional group on the surface. The different surface modifications are as follows: 1. Reactive organo‐silane surfaces are widely used sensor surfaces for modifcation Metal oxide surfaces were silanized followed by linking chemical groups
  • 42. Cyanuric chloride activation on hydroxyl surfaces
  • 43. Covalent attachment on acrylic surfaces (plastic)
  • 44. Activation of carboxy surfaces by DCC
  • 45. Eg., Modification of surfaces (sensor/biomolecule) through carbodiimide, avidin-biotin interactions
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  • 48. a.-OH surfaces are first modified with carbonyldiimidazole (CDI) to formreactive intermediate- formsa stable carbamatebond to anaminoterminated biomolecule b.-NH2 gp can be modified with glutaraldehyde which forms imine bond (Schiff's base) with an aldehyde, leaving theother aldehydefree for repeating this chemistry with an amino-terminated molecule c.Symmetric diisothiocyanates have also been used as bifunctional linkers for attachment of amine-functionalized surfaces to either thiol-terminated or amine-terminated molecules d.Heterobifunctional linker sulfosuccinimidyl-4-(N- maleimidomethyl)-cyclohexane-1-carboxylate, which first reacts with surface-bound -NH2 through displacement of the NHS, subsequently, a thol-tethered molecule can be reacted with the pendant maleimide gp. Toform surface- conjugated molecules e. EDC/NHS reaction - the most commonones
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  • 63. ImmobilizationTechnology Must be oriented in a specific direction ‐ immobilization technology Five major immobilization methods used in the preparation of Chemical and bio‐ sensors. 1.Covalent binding ‐ attachment of the active component to the transducer surface using a chemical reaction such as peptide bond formation or linkage to activated surface groups (thiol, epoxy, amino, carboxylic, etc) 2. Entrapment ‐ physical trapping of the active component into a film or coating. 3.Cross‐linking ‐ similar to entrapment, only a polymerization agent (such as gluteraldehyde) is used to provide additional chemical linkages between the active, entrapped component and the film or coating. 4.Adsorption ‐ association of the active component with a film or coating through hydrophobic, hydrophilic, and/or ionic interactions. 5.Biological binding ‐ association of an active biomolecule to a film or coating through specific, biochemical binding
  • 64. The covalent binding method is based on the binding of enzymes and water‐insoluble carriers by covalent bonds. The functional groups that may take part in this binding are listed below: Amino group Carboxyl group Sulfhydryl group, Hydroxyl group Imidazole group Phenolic group Thiol group Threonine group Indole group Covalent attachment to a support matrix must involve only functional groups of the enzyme that are notessentialfor catalytic action. Higher activities resultfrom prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised: Covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate. 1. A reversible, covalently linked enzyme-inhibitor complex. 2. A chemically modified soluble enzyme whose covalent linkage to the matrix is achieved by newly incorporated residues. 3. A zymogen precursor. Zymogen: Any of a group of compounds that are inactive precursors of enzymes and require some change (such as the hydrolysis of a fragment that masks an active enzyme) to become active
  • 65. Hence, covalent binding can be brought about by the following: Diazotization : Amide bond formation : Alkylation and Arylation: SUPPORT--N=N--ENZYME. SUPPORT--CO-NH--ENZYME SUPPORT--CH2-NH-ENZYME SUPPORT--CH2-S--ENZYME SUPPORT--CH=N--ENZYME SUPPORT--CNH-NH--ENZYME SUPPORT--S-S--ENZYME Schiff's base formation : Amidation reaction : Thiol-Disulfide interchange : UGI reaction Gamma-Irradiationinduced coupling Carrier binding with bifunctional reagents : SUPPORT-O(CH2)2N=CH(CH2)3 CH=N-ENZYME
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  • 68. Glutaraldehyde- The surfaces (proteins) of microorganisms are linked with the surfaces of other microorganisms by aldehyde groups of glutaraldehyde
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  • 71. Entrapmentand encapsulation The immobilized enzyme can be classified into four types: particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of handling and ease of application. Particles - The particle form of immobilized enzyme on solid particles. Membranes - Enzyme membranes can be prepared by attaching enzymes to membrane- type carriers, or by molding into membrane form. The molding is done after the enzymes have been enclosed within semi-permeate membranes of polymer by entrapment. Tubes- Enzyme tubes are produced using Nylon and polyacrylamide tubes as carriers. The polymer tube isfirst treated ina seriesof chemicalreactionsand theenzymeisboundby diazo coupling to give a tube in a final step. Fibers - Enzymes that have been immobilized by entrapment in fibers to form enzyme fibers. The solid supports used for enzyme immobilization can be inorganic or organic . Some organic supportsinclude: P olysaccharides, Proteins, Carbon, P olystyrenes, P olyacrylates, MaleicAnhydride basedCopolymers,Polypeptides,Vinyl andAllyl Polymers,and Polyamides.
  • 72. ImmobilizationthroughEntrapment The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into lattice and micro capsule types
  • 73. Cross‐linking is based on the formation of chemical bonds, as in the covalent binding method. The immobilization is performed by the formation of intermolecular cross‐ linkages between the enzyme molecules by means of bi or multifunctional reagents. The most common reagent used for cross‐linking is glutaraldehyde. Cross‐linking reactions are carried out under relatively severe conditions. These harsh conditions can change the conformation of active center of the enzyme; and so may lead to significant loss of activity. The carrier‐binding method is the oldest immobilization technique for enzymes. In this method, the amount of enzyme bound to the carrier and the activity after immobilization depend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier: The selection of the carrier depends on the nature of the enzyme itself, as well as the: Particle size Surface area Molar ratio of hydrophilic to hydrophobic groups Chemical composition ‐polysaccharide derivatives such as cellulose, dextran, agarose, and polyacrylamide gel.
  • 74. Physical Adsorption Mode Based on the physical adsorption of enzyme protein on the surface of water-insoluble carriers. Theprocessesavailable for physical adsorption of enzymesare: StaticProcedure Electro-deposition Reactor Loading Process Mixing or Shaking Bath Loading Adsorption tendsto be lessdisruptive to theenzymaticprotein thanchemicalmeansof attachment because the binding is mainly by hydrogen bonds, multiple salt linkages, and Van der Waal's forces. Becauseof theweak bondsinvolved,desorption of theprotein resulting from changesin temperature, pH,ionicstrengthor eventhemerepresence of substrate, is often observed. Another disadvantage is non-specific, further adsorption of other proteins or other substances as theimmobilized enzymeisused.Thismayalter the properties of the immobilized enzyme or, if thesubstanceadsorbed isa substratefor theenzyme, the rate will probably decrease depending on the surface mobility of enzyme and substrate. Adsorption of the enzyme may be necessary to facilitate the covalent reactions described later inthispresentation. Stabilization of enzymestemporarily adsorbed ontoa matrix hasbeen achieved by cross-linkingtheprotein in a chemical reaction subsequent to its physical adsorption.
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