![Page 1: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/1.jpg)
Biosensor: surface functionalisation
Integrating the biorecognition elements
Dr. MAK Wing Cheung (Martin)
(6 Feb 2015)
Biosensors & Bioelectronic Centre, IFM
Email: [email protected]
Phone: +4613286921
![Page 2: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/2.jpg)
Physico-chemical imobilisation
Adsorption
Entrapment
Cross-linking
Chemical modification
Self assembling
Covalent modification
Affinity assembling
Outline
![Page 3: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/3.jpg)
How to couple the bio-recognition element to the transducer surface?
Bioreceptors and transducers
![Page 4: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/4.jpg)
Immobilisation
Why we need immobilization?
![Page 5: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/5.jpg)
Immobilisation methods
- Adsorption
- Covalent bond
- Crosslinking
- Entrapment
- Encapsulation
![Page 6: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/6.jpg)
Simple
Complex
Comparison of immobilisation methods
Less stable
More stable
![Page 7: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/7.jpg)
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)
![Page 8: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/8.jpg)
Gel entrapment
Gel entrapment
Hydrogel: is a network of polymer chains that are hydrophilic
(high water content).
![Page 9: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/9.jpg)
• 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
![Page 10: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/10.jpg)
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
![Page 11: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/11.jpg)
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?
![Page 12: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/12.jpg)
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
![Page 13: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/13.jpg)
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
![Page 14: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/14.jpg)
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…..
![Page 15: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/15.jpg)
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.
![Page 16: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/16.jpg)
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
![Page 17: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/17.jpg)
Functional group of proteins
COOH NH2
OH SH
Protein
![Page 18: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/18.jpg)
Bi-functional linker (BFL)
http://www.piercenet.com/cat/crosslinking-reagents
Two chemical functional groups can be covalently cross-linked with bi-functional linker
![Page 19: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/19.jpg)
Chemistry for immobilisation Amine chemistry
Thiol chemistry Carboxyl chemistry
![Page 20: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/20.jpg)
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
![Page 21: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/21.jpg)
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
![Page 22: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/22.jpg)
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
![Page 23: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/23.jpg)
Supramolecular approach
Host-guest complex
Host molecule Guest molecule
![Page 24: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/24.jpg)
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
![Page 25: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/25.jpg)
Can you implied what are the immobilisation methods?
What are the advantages and dis-advantages?
Discussion
Entrapment
Covalent bond Physical adsorption
![Page 26: Biosensor: surface functionalisation Integrating the ...€¦ · Biosensor: surface functionalisation Integrating the biorecognition elements Dr. MAK Wing Cheung (Martin) (6 Feb 2015)](https://reader035.vdocument.in/reader035/viewer/2022062415/605ea62c317f854b9f3c8367/html5/thumbnails/26.jpg)
Please contact me if you have more questions….
Dr. Wing Cheung Mak (Martin)
Email: [email protected]
Phone: +4613286921
Office: 2E:665
The end