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Biosensor: surface functionalisation
Integrating the biorecognition elements
Dr. MAK Wing Cheung (Martin)
(6 Feb 2015)
Biosensors & Bioelectronic Centre, IFM
Email: [email protected]
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: [email protected]
Phone: +4613286921
Office: 2E:665
The end