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. Active biological receptor in aqueous environment 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 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.

(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.

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

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

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

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

Immobilisation de biomolécules sur des polymères via des ponts avidine-biotine Capteur enzymatique enzyme oligonucléotide anticorps Puce à ADN 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 Graphite-NTA-Ni AChE - (His)6 AChE Ni Ni G R A P I T 2 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

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)

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

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

Principle of Concanavalin A AChE G R A P H I T E 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 SH Thiols dissolved in ethanol Surface dipped into solution Self assembled monolayer

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

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