2 Récepteur biologique

4 Transduction

1 composé à analyser

3 Méthode d’immobilisation Type de surface

5 Processeur

BIOCAPTEUR : éléments

IMMOBILISATION METHODS

Bioreceptor immobilisation at surfaces: not trivial

1. Active biological receptor in aqueous environment

2. Proteins can denature and loose recognition/catalytic ability at (transducer) surfaces

1. 2.

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)

2.1 Covalent bondingChemical 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 - Chemical attachment

2.2 CrosslinkedIn 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)

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.

CH2

CH

O

CH2

CH

O

NH2 NH2. . . .

CH2

CHHO

NH NH

CH2

CHHO

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

NH2NH2 NH2NH2. . . .

CH2

CHHO

CH2

CHHO

NHNH NHNH

CH2

CHHO

CH2

CHHO

Example: protein immobilisation through surface lysine

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.

Covalent immobilisation of oligonucleotides: DNA/RNA

CH2

CH

O

CH2

CH

O

NH2 NH2. . . .

CH2

CHHO

NH NH

CH2

CHHO

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

CH2

CH

O

NH2NH2 NH2NH2. . . .

CH2

CHHO

CH2

CHHO

NHNH NHNH

CH2

CHHO

CH2

CHHO

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

3.1 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 - Non-covalent attachment

3.3 Biological interactions: affinity

Taking advantage of strong and specific biological interactions between proteins and ligands.

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.

NH3+ NH3

+ NH3+ NH3

+ NH3+ NH3

+

- - -- - - - - -

- - -NH3+ NH3

+ NH3+NH3

+NH3+ NH3

+NH3+ NH3

+NH3+ NH3

+ NH3+ NH3

+NH3+NH3+ NH3

+NH3+ NH3

+NH3+

- - -- - -- - -

- - - - - -- - -- - -

- - -

Oligonucleotides contain negatively charged phosphate goups in their back bones. These can form electrostatic bonds with positively charged amine groups on surfaces.

Electrostatic interactions

Attractive interactions between opposite charges on biological receptor molecule and transducer surface.

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

2. 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

Avidin/biotin: strongest known biological interaction

Biological receptor

Biotin

Avidin (tetrameric protein)

Biotin

transducer

1) Covalent attachment of biotin to transducer and bioreceptor2) Self assembly to form ‘sandwich’:

Antigen/antibody:Antigen coupled to biological receptor, antibody on surface

Biological receptor

Antigen (e.g. small protein)

Antibody

transducer

Specific coupling via biological affinity

Immobilization of biomolecules by affinity interactions (avidin-biotin)

biotin avidin

avidin-biotin complex

S

NHHN

O

(CH2)4 CO

(CH2)n N

electropolymerizable biotin

+1015 M-1

association constant

Immobilisation de biomolécules sur des polymères via des ponts avidine-biotine

enzyme

oligonucléotide

anticorps

Capteur enzymatique

Immunocapteur

Puce à ADN

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:

- a histidine tail in the enzyme molecule- a support containing a metal chelate

The presence:

Functionalisation of the electrode surface with a metal chelate

Utilisation of a genetically modified AChE to incorporate a six-histidine tail - AChE -(His)6

Ni

CH

CO

O

N

CH2 CO

O

O OH2

CH2

OC

OH2

O

GRAPHITE

Graphite-NTA-Ni

- (His)6AChE

HN

AChE

N

Ni

CH

CO

O

O

N

CH2 CO

O

O OH2

CH2

OC

GRAPHITE

Principle of MCA

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

Conclusion:

Higher sensitivity compared to physical entrapment

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

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

G

R

A

P

H

I

TE

Sugar

Concanavalin A

AChE

AChE

Principle of 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 containing proteins in ECM (fibronectin) RGD peptides

Integrin (adhesion factor)

Cell with surface integrins

Extra cellular matrix (ECM)

Biomaterial surface

RGD/cells: RGD is a tri-peptide (Arg-Gly-Asp) that promotes cell binding

(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

Gold/sulphur bond

Hydrophobic interactions

Self Assembled Monolayers

Thiols dissolved in ethanol

Surface dipped into solution

Self assembled monolayer

Functional head group:OH, NH2, COOH

Alkane

SH

Surface Modification: silanes on metal oxides

C2H5O C2H5OC2H5O

+ H 2O +

X

C2H5O C2H5OC2H5O

+ H 2O +

O O O O

Hydrolysis

Condensation

Glass (SiO2)Or other metal oxide

O O O

Functional head group

Alkane

Si

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