Biosensor: surface functionalisation

Integrating the biorecognition elements

Dr. MAK Wing Cheung (Martin)

(6 Feb 2015)

Biosensors & Bioelectronic Centre, IFM

Email: mamak@ifm.liu.se

Phone: +4613286921

Physico-chemical imobilisation

Adsorption

Entrapment

Cross-linking

Chemical modification

Self assembling

Covalent modification

Affinity assembling

Outline

How to couple the bio-recognition element to the transducer surface?

Bioreceptors and transducers

Immobilisation

Why we need immobilization?

Immobilisation methods

- Adsorption

- Covalent bond

- Crosslinking

- Entrapment

- Encapsulation

Simple

Complex

Comparison of immobilisation methods

Less stable

More stable

Physical immobilisation method

Physical immobilisation methods including adsorption, entrapment

and encapsulation

Advantages: - Simple and easy to perform

- Mind conditions

- Minimum modification to biomolecules (preserve function)

Disadvantages: - Less stable (leakage from matrix)

- Limited to immobilisation on hydrophilic surface

- Diffusion barrier by the matrix (e.g. gel)

Gel entrapment

Gel entrapment

Hydrogel: is a network of polymer chains that are hydrophilic

(high water content).

• Hydrophobic forces - Hydrophobic groups intended to interact with hydrophobic surface to minimize

the surface energy

- Strong force

• Electrostatic forces - Attraction between opposite charges

- Strong force

• Van der waals forces - Fluctuations in electron clouds around molecules oppositely polarized

neighboring atoms

- Weak force

• Hydrogen bonds - Hydrogen shared between electronegative atoms (N, O)

- Stronger than Van der waals force

Driving forces for physical adsorption

Electrostatic self-assembly

Cationic polyelectrolytes

Anionic polyelectrolytes

- Simple

- Nanoscale thickness (thickness controlled by layer number(s))

- Integration of multiple biological function with desired fashion

Layer-by-Layer (LbL) technology

Properties of amino acids

e.g. hydrophobic domain e.g. hydrophilic domain (i.e. charged group)

Question: What are the forces contributed for physical immobilisation?

https://www.youtube.com/watch?v=hafoobuzQYs#t=44

Immobilisation in practice (electrode)

Immobilized enzyme onto electrode

surface (gel entrapment)

High precision and high throughput

immobilisation

https://www.youtube.com/watch?v=Lt9KgVt_m6s

Immobilisation in practice (membrane)

Iso-flow disperser

High-throughput immobilization biomolecule

onto membrane surface (0.5 meter per minute)

Contact immobilization method

(nozzle in contact with membrane)

Nozzle

Test line Control line

Nitrocellulose membrane

Why physical adsorption?

Discussion: Why most of the commercial medical biosensors use physical

adsorption method for immobilisation of biomolecules?

- Cost

- Single use

- Contamination with biological fluid sample

- Others…..

Chemical immobilisation method

Chemical immobilisation methods including covalent binding and

crosslinking

Advantages: - More stable

- Allow immobilisation to wide range of surfaces

(e.g. metal, semi-conductor, polymer etc.)

- Allow to immobilize biomolecules in a more

controlled way (better reproducibility).

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.

Journal of Industrial Microbiology, 9 (1992) 163-172

Why chemical immobolisation

Analytes

Biosensors for process monitoring Relatively long processing time

(Need stable biosensors)

Beer fermentation process

- Real time quality control

- Better taste

Functional group of proteins

COOH NH2

OH SH

Protein

Bi-functional linker (BFL)

http://www.piercenet.com/cat/crosslinking-reagents

Two chemical functional groups can be covalently cross-linked with bi-functional linker

Chemistry for immobilisation Amine chemistry

Thiol chemistry Carboxyl chemistry

Receptors immobilisation

Optimal orientation of receptors (antibodies) results in:

• Greater affinity of the antigen

• Increased sensitivity of the affinity biosensors

• High reproducibility of the immobilisation

Early methods of immobilisation using the NH2 and COOH groups of the

antibody for covalent attachment. These groups, being distributed throughout the

molecule surfaces generated random orientation and low affinity. This is

particularly critical if NH2 groups are used for the immobilisation because they

are preferentially located in the recognition site.

Amino linking of antibodies

Inactive Active

OR

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).

Bioaffinity assembly

Supramolecular approach

Host-guest complex

Host molecule Guest molecule

Protein-A immobilisation

Protein A is a 56 kDa surface protein originally found in the cell wall of

the bacterium Staphylococcus aureus. It binds the heavy chain within the

Fc region of most immunoglobulins.

Can preserve the Fab region of the antibodies free for antigen binding

Antibodies immobilization via Protein A

Antibody

Protein A

Can you implied what are the immobilisation methods?

What are the advantages and dis-advantages?

Discussion

Entrapment

Covalent bond Physical adsorption

Please contact me if you have more questions….

Dr. Wing Cheung Mak (Martin)

Email: mamak@ifm.liu.se

Phone: +4613286921

Office: 2E:665

The end