BIOCAPTEUR : éléments
1 composé à analyser
2 Récepteur biologique
5 Processeur
4 Transduction
3 Méthode d’immobilisation
Type de surface
IMMOBILISATION METHODS
Bioreceptor immobilisation at surfaces:
not trivial
1.
2.
1. Active biological receptor in
aqueous environment
2. Proteins can denature and
loose recognition/catalytic
ability at (transducer)
surfaces
1 - Physical ‘entrapment’
1.1 Micro-encapsulation
The biological receptor is entrapped behind a permeable
membrane that allows small molecules (analytes, inorganic
ions, etc.) pass freely while the biological receptor is
contained near the transducer surface.
1.2 Entrapment
A crosslinked polymer network prepared in the presence of
biological receptor and thus incorporated into the pores of
the polymer structure.
Entrapment in PVA-SbQ
Immobilisation in SOL-GEL
(Dis)Advantages of physical entrapment
+ Does not interfere with bioreceptor reliability
+ Limits contamination by proteins in sample
+ Limits biodegradation of receptor
- Diffusion of analytes to and from the biological receptor
can be slow
- Entrapment of undesired (interfering) molecules behind
membrane/ inside polymer network
- Leakage of bioreceptor (can be avoided by chemical
crosslinking)
2 - Chemical attachment
2.1 Covalent bonding
Chemical bond between a chemical group on the biological receptor
and a chemical group on the transducer surface. The chemical
reaction must work under conditions that are compatible with
integrity of the bioreceptor (aqueous, low temperature, non extreme
pH or ionic strength…)
2.2 Crosslinked
In this method, the bioreceptor molecules are linked to each other as
well as to the transducer surface in a crosslinked polymer network
using bi-functional monomers such as glutaraldehyde.
Functional groups on biomolecule surface:
proteins
Enzymes, antibodies (and of course receptor proteins) are all
proteins
A number of their amino acid building blocks have functional side
chains that can be used for chemical attachment
Amino acids with functional groups:
Lysine (NH2), Cysteine (SH), Serine (OH), Aspartic Acid (COOH)
Example: protein immobilisation through
surface lysine
NH
..2
NH
.. 2
CH2
O
CH2
NH
NH
CH2
CH2
O
CH
CH
HO
CH
HO
CH
Proteins contain primary amine groups on lysine residues. The
lone pair electrons (double dots) attack the electrophilic
carbon on the epoxide group, forming a covalent bond between
the protein and the substrate.
Covalent immobilisation of oligonucleotides:
DNA/RNA
Single stranded
oligonucleotides
contain primary amine
groups on the A, G,
and C residues. The
amine groups attack
the carbon on the
epoxide group and
form a covalent bond.
Monomers that are involved in
chemical attachment are not available
for binding to nucleotides.
NH
..2
NH
.. 2
CH2
O
CH2
NH
NH
CH2
CH2
O
CH
CH
HO
CH
HO
CH
(Dis)Advantages of covalent attachment
+ Enhanced stability
+ When using covalent attachment good control over
biological receptor orientation is possible
+ When using electrode as transducer can use a
electronically conducting linker giving very efficient
translation of biorecognition to electronic.
-
-
Damage to the biological receptor and loss of
selectivity/catalytic activity due to chemical binding event
(especially in crosslinking)
Mechanical strength of system can be poor
3 - Non-covalent attachment
3.1 Electrostatic interactions
Between charged groups on the biological receptor and oppositely
charged groups on the transducer surface. These are mainly used for
immobilisation of DNA.
3.2 Physical adsorption to the surface
Many materials (e.g. glass, gold, silica gel) adsorb proteins on their
surfaces. No reagents are required in this method. Proteins usually
loose their 3D structure and biological recognition ability.
3.3 Biological interactions: affinity
Taking advantage of strong and specific biological interactions between
proteins and ligands.
Electrostatic interactions
Attractive interactions between opposite charges on biological receptor
molecule and transducer surface.
-
Oligonucleotides contain negatively
charged phosphate goups in their
back bones. These can form
electrostatic bonds with positively
charged amine groups on surfaces.
-
-
NH3+
-
NH3+
NH3+
- 3+
NH
-NH3+
NH-3+
Physical adsorption
•
•
•
Through Van der Waals interactions
Only useful for short term attachment of biological receptors
Or as pre-coatings for cell attachment:
1.
Pre-adsorption of extracellular matrix proteins to either enhance
(fibronectin) cell attachment
Albumin is generally used to ‘passivate’ surfaces: a monolayer of
albumin prevents further adsorption of proteins.
2.
Fibronectin coated
Albumin coated
Specific coupling via biological affinity
1) Covalent attachment of biotin to transducer and bioreceptor
2) Self assembly to form ‘sandwich’:
Biological receptor
Biotin
Avidin (tetrameric protein)
Biotin
transducer
Avidin/biotin:
strongest known biological interaction
Specific coupling via biological affinity
Biological receptor
Antigen (e.g. small protein)
Antibody
transducer
Antigen/antibody:
Antigen coupled to biological receptor, antibody on surface
Immobilization of biomolecules by affinity
interactions (avidin-biotin)
O
HN
NH
S
(CH2 )4 C (CH2 )n N
O
electropolymerizable biotin
biotin
avidin-biotin complex
avidin
+
association constant
1015 M-1
Immobilisation de biomolécules sur des
polymères via des ponts avidine-biotine
Capteur enzymatique
enzyme
oligonucléotide
Puce à ADN
anticorps
Immunocapteur
Immobilisation de plusieurs couches d ’enzyme
Principle of MCA
• Ability of certain metal ions such as Ni2+, Cu2+, Zn2+ to
bind strongly and reversibly to enzymes containing
histidine or cysteine tails in the proteine sequence
Conditions:
The presence:
- a histidine tail in the enzyme molecule
- a support containing a metal chelate
 Utilisation of a genetically modified AChE
to incorporate a six-histidine tail - AChE -(His)6
 Functionalisation of the electrode surface with a
metal chelate
Principle of MCA
CO
G
R
A
P
H
I
T
E
CO
O
CH2
CO
AChE - (His)6
CH
O
O
N
Ni
CH2
OC
OH2
O
G
R
A
P
H
I
T
E
O
CH2
CH
O
O
N
Ni
CH2
OC
OH2
O
OH2
Graphite-NTA-Ni
CO
N
HN
AChE
Immobilisation steps

Synthesis of the nitrilotriacetic acid (NTA) (Hochuli et al. 1987)

Functionalisation of the graphite with hydroxyls groups;
activation of the –OH groups
 Charging

of the activated graphite with the metal chelate
Complexation with Ni2+ ions

Electrode manufacturing (deposition of the functionalised
graphite by screen-printing)
 Enzyme immobilisation
Comparison with other methods
Characteristics
Sensitivity (mA/M)
Linear range (M)
AFFINITY
PHYSICAL
ENTRAPMENT
3
0.16
1 10 –6 – 6 10
–5
1 10 –5 – 4 10
Conclusion:
 Higher
sensitivity compared to physical
entrapment
–4
Principle of Concanavalin A
• Ability of concanavalinA to bind strongly and reversibly
to enzymes containing sugars
Conditions:
The presence:
- a glycalated enzyme
- a support containing concanavalin A
 Functionalisation of the electrode surface
with a sugar or concanavalin A
Principle of Concanavalin A
G
R
AChE
A
P
H
I
T
E
AChE
Sugar
Concanavalin A
Immobilising cells through biological affinity
Cell surfaces are decorated with proteins
Some of these (integrins) are responsible for cell attachment to the
extracellular matrix (ECM)
Short peptide sequences frequently found in ECM proteins can be
immobilised on synthetic surfaces
Resulting in highly specific cell immobilisation
Most well known example is fibronectin tri-peptide RGD (Arg-Gly-Asp)
RGD/cells: RGD is a tri-peptide (Arg-Gly-Asp) that
promotes cell binding
Cell with surface integrins
Extra cellular matrix (ECM)
Biomaterial surface
RGD containing proteins in ECM (fibronectin)
RGD peptides
Integrin (adhesion factor)
(Dis)Advantages of non-covalent attachment
+ Electrostatic interactions have been used with
much success for immobilisation of DNA for gene
chips
+ Biological interactions: strong and highly
selective
+ Immobilisation under very mild conditions
(buffer)
- Susceptible to changes in pH, temperature, ionic
strength.
- Mechanical strength of system can be poor
How to functionalise and pattern transducer
surfaces: Functionalisation and patterning
• Self assembled monolayers: thiols on gold
• Silanes on metal oxides
Self-assembled Monolayers (SAMs)
• organic, highly oriented surfaces
• formed by adsorption of alkanethiols, X(CH2)nSH,
onto gold
Hydrophobic interactions
Gold/sulphur bond
Self Assembled Monolayers
Functional head group:
OH, NH2, COOH
Alkane
Thiols dissolved in ethanol
SH
Surface dipped into solution
Self assembled monolayer
Surface Modification:
silanes on metal oxides
Functional
head group
Alkane
X
Hydrolysis
+ H 2O +
Condensation
Si
O
C2H5O
C H O
C 2H 5 O 2 5
Glass (SiO2)
Or other metal
oxide
O
O
O
O O
O
Comparing surface functionalisation methods
Thiol/gold system:
very high level of order achieved
precise control of direction and density
Useful for electronic and some optical sensors, not for fluorescence
Straightforward patterning using UV/ micro contact printing
Silane/metal oxide:
More challenging to obtain a homogeneous monolayer
(not a self assembly process)
More generally useful as it works on all metal oxide surfaces