1. BIOCAPTEUR : éléments 1 composé à analyser 2 Récepteur biologique
5 Processeur
4 Transduction
3 Méthode d’immobilisation
Type de surface

2. IMMOBILISATION METHODS

3. Bioreceptor immobilisation at surfaces: not trivial
Active biological receptor in aqueous environment
Proteins can denature and loose recognition/catalytic ability at (transducer) surfaces

4. 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.

5. Entrapment in PVA-SbQ

6. Immobilisation in SOL-GEL

7. (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)

8. 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.

9. 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)

10. Example: protein immobilisation through surface lysine
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.

11. 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.

12. (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

13. 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.

14. 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.

15. Physical adsorption Through Van der Waals interactions
Only useful for short term attachment of biological receptors
Or as pre-coatings for cell attachment:
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.
Fibronectin coated Albumin coated

16. Specific coupling via biological affinity
Covalent attachment of biotin to transducer and bioreceptor
Self assembly to form ‘sandwich’:
Biological receptor
Biotin
Avidin (tetrameric protein)
transducer
Avidin/biotin:
strongest known biological interaction

17. Specific coupling via biological affinity
Biological receptor
Antigen (e.g. small protein)
Antibody
transducer Y Y
Antigen/antibody:
Antigen coupled to biological receptor, antibody on surface

18. Immobilization of biomolecules by affinity interactions (avidin-biotin)H O ( C 2 ) 4 n
electropolymerizable biotin
biotin
avidin
avidin-biotin complex
association constant +
1015 M-1

19. Immobilisation de biomolécules sur des polymères via des ponts avidine-biotine
Capteur enzymatique
enzyme
oligonucléotide
anticorps
Puce à ADN
Immunocapteur

20. Immobilisation de plusieurs couches d ’enzyme

21. 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

22. Principle of MCA Graphite-NTA-Ni AChE - (His)6 AChE Ni Ni G R A P I T2 CO OC HN
AChE N Ni C H O 2 CO OC G R A P I T E O G R A P H I T E
- (His)6
AChE Ni
Graphite-NTA-Ni

23. Immobilisation steps
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
Enzyme immobilisation
Synthesis of the nitrilotriacetic acid (NTA) (Hochuli et al. 1987)
Electrode manufacturing (deposition of the functionalised graphite by screen-printing)

24. Comparison with other methods
Characteristics
AFFINITY
PHYSICAL ENTRAPMENT
Sensitivity (mA/M) 3
0.16
Linear range (M)
1 10 –6 – 6 10 –5
1 10 –5 – 4 10 –4
Conclusion:
Higher sensitivity compared to physical entrapment

25. Principle of Concanavalin A
Ability of concanavalinA to bind strongly and reversibly to enzymes containing sugars
Conditions:
- a glycalated enzyme
- a support containing concanavalin A
The presence:
Functionalisation of the electrode surface with a sugar or concanavalin A

26. Principle of Concanavalin A
AChE G R A P H I T E
Sugar
Concanavalin A

27. 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)

28. 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)

29. (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

30. How to functionalise and pattern transducer surfaces: Functionalisation and patterning
Self assembled monolayers: thiols on gold
Silanes on metal oxides

31. Self-assembled Monolayers (SAMs)
organic, highly oriented surfaces
formed by adsorption of alkanethiols, X(CH2)nSH, onto gold
Hydrophobic interactions
Gold/sulphur bond

32. Self Assembled Monolayers
Functional head group:
OH, NH2, COOH
Alkane SH
Thiols dissolved in ethanol
Surface dipped into solution
Self assembled monolayer

33. Surface Modification: silanes on metal oxides
Functional head group
Alkane Si X
Hydrolysis
Condensation
+ H
+ H
O +
O + 2 2
O O O O C C H H O O C C H H O O
Glass (SiO2)
Or other metal oxide 2 2 5 5 C C H H O O 2 2 5 5 2 2 5 5
O O O

34. 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