biosensor: surface functionalisation integrating the ... · schematic representations of igg...

Biosensor: surface functionalisationIntegrating the biorecognition elements

Valerio.beni@liu.se2014/02/07

Outline

� Physico-chemical imobilisation

� Adsorption

� Entrapment

� Cross-linking

� Chemical modification

� Self assembling

� Covalent modification

� Affinity assembling

Of what we will talk?

Main elements of a biosensor.

(a) Biorecognition element

(b) transducer

(c) amplifier

(d) Signal converter

(e) recorder

Popular approaches for the immobilisation of biorecognition elements

• Adsorption

• Entrapment

• Crosslinking

• Encapsulation

• Covalent binding

Bio-molecules immobilisation

Easy to perform

Complexto perform and

time consuming

Physico-chemical Adsorption

Surface self assembling of bio-molecules Adsorption

Advantages: Easy and cost effective approach, less destruptive (biorecognition element mantain their function.

Problems: low controll of molecules orientation, less stable.

Forces governing adsorption

Limitations

+ +- -

Poor stability of the modified layer

Poor orientation of the biorecognition elements

Entrapment of biomolecules

• Membranes -Enzyme membranes can be prepared by attaching enzymes to

membrane-type carriers, or by molding into membrane form.

Example is the entrapping of enzymes in nafion membrane.

Electrostatic self assembly

Palyelectrolyte –The enzymes are trapped in between sequencial layers of charged polymers.

Electrostatic self assembly – protein

multilayers

Colorimetric detection of Glucose via the use of Sequential Enzymatic Reaction

Glucose oxidase

Peroxidase

DA67: Chromogenfor enzymatic reaction transduction

Entrapping biomolecules by electropolymerisation

Possible monomers

Entrapping in hydrogels

Hydrogel: is a network of polymer chains that are hydrophilic; this contains high quantity of water.

Cross-linking

Cross-linking of the enzymes ensure higher stabilityand robustness to the sensor.

Cross-linking is obtained by using molecules that are able to interact with the aminoacidic residues and to bridge two neigbouring enzymes..

Covalent binding

Chemical modification

Advantages:

• More stable

• Allow immobilisation to a wider range of surfaces (e.g. metal, semi-conductor, polymer etc.).

• Allow to immobilise biomolecules in a more controlled way (better orientation, better reproducibility).

• Allow to create suitable environment for:

- Biomolecules immobilisation

- Reduction of non-specific interaction

Disadvantages:

• Required chemical modification of the biomolecules.

• Harsh chemical reaction may affect the function of the biomolecules.

• Time consuming and increased complexity in sensor preparation.

Metal surface functionalisationSelf assembling monolayer (SAM)

Spontaneous but ordered assembling of molecules.

Most used is the self assembling of alkane thiols(R-SH).

This approach allow to create stable intermediate layer for the controlled immobilisation of biomolecules onto metals (Au, Cu, Ag)…

Si Self-Assembled Monolayers (SAMs)

Equivalent to Thiol SAM but:

More stableSuitable for glass, quartz, Pt and other semiconductors where thiols do not attach.

Polymeric substrate

Electrode

Covalent Binding

The covalent binding method is based on the binding of biomolecules 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 biomolecule that are not essential for catalytic/recognition activity.

Functional group of proteins

Covalent binding (EDC/NHS)

Bio-affinity assembling

Streptavidin: is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Its homo-tetramers have an extraordinarily high affinity for biotin. Dissociation constant (Kd) on the order of ≈10-14 mol/L.

Biotin: also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin (vitamin B7).

Schematic representations of IgG

immobilization on a gold surface

modified by various cysteine-tagged

protein G constructs. Anal. Chem.,

79 (7), 2680 -2687, 2007.

Schematic representations of

DNA-directed antibody

immobilization by protein G-

DNA conjugate. Anal. Chem.,

79 (17), 6534 -6541, 2007

(a) Fluorescence response to different concentrations of CA 19-9 antigen (b) Schematic illustration of the

sandwich immunoassay configuration used. (c) Fluorescence intensity as a function of the CA 19-9 concentration

in the samples. (d) Specificity of the antibody array. Antibodies for CA 19-9 and CEA were spotted onto the array,

and samples containing either CA 19-9 or CEA were added. J. Am. Chem. Soc., 128 (3), 676 -677, 2006

Supramolecular approach

Summary

• Different approaches for the immobilisation of the biorecognitionelement onto the transducer are possible.

• Degree of complexity increases with the increase in the control of the final surface.

• Chemical, biological and supramolecular interaction can be used to functionalise surfaces.

• Recognition ability of biorecognition element can strongly influenced by the immobilisation process.