Lecture FO7 Affinity biosensors

Transcription

1 Lecture FO7 Affinity biosensors Dr. MAK Wing Cheung (Martin) Biosensors & Bioelectronic Centre, IFM Phone: (21 Feb 2014)

2 Affinity biosensors Affinity biosensors: devices in which bio-recognition molecules bind analyte molecules leading to formation or dissociation of complex, causing a physicochemical change that is detected by a transducer Receptor molecules: i) antibodies ii) DNA iii) receptors

3 Different between affinity and enzyme biosensors

4 Design parameters for affinity biosensors

5 Enzyme Linked ImmunoSorbent Assay (ELISA) 1) Immobilization of capture antibodies 2) Addition of samples 3) Addition of detection antibodies (with an enzyme label) 4) Addition of enzyme substrate for colour development 5) Measure by optical transducers

6 Direct affinity assay (sandwich assay) Used to detect the presence of antigens in a sample Signal directly proportional to analyte concentration Only for large molecular weight analyte Discussion: Why sandwich assay cannot be used for small molecular weight analyte?

7 Why the products are coloured? (Artificial substrate e.g. 3,3,5,5 -Tetramethylbenzidine) Procedures for direct affinity assay Immobilization of primary antibodies Y Y Y Washing steps (remove unbounded reagents) Block the empty surface with bovine serum albumin (BSA) Y Y Y Washing steps Addition of sample analytes Y Y Y Washing steps Addition of secondary labelled antibodies (enzyme label) Y Y Y Y Y Y Y Addition of enzyme substrate Y Y Y Y Y Measure colour intensity substrate Product (with colour) Spectrophotometer (optical transducer)

8 Signal intensity Interpretation of direct affinity assay Case I : Sample with high concentration of analytes substrate Y Y Y Y Y Y Product (with colour) High signal intensity Case II : Sample with moderate concentration of analytes substrate Y Y Y Y Product (with colour) Moderate signal intensity Case III : Sample without analyte Y Y Y Low signal

9 Competitive affinity assay Labelled analyte (labelled with enzyme) Analyte from sample Analytes presence in sample and labelled analytes are competing for antibodies binding sites Signal reversely proportional to analyte concentration Suitable for small molecular weight analyte Lower sensitivity compare with direct assay

10 Procedures for competitive affinity assay Immobilization of primary antibodies Addition of sample analytes and labelled analytes (compete for antibodies binding sites) Y Y Y Washing steps (remove unbounded reagents) Block the empty surface with bovine serum albumin (BSA) Y Y Y Washing steps Y Y Y Addition of enzyme substrate Y Y Y Washing steps substrate Product (with colour) Measure colour intensity Spectrophotometer (optical transducer)

11 Signal intensity Interpretation of competitive affinity assay Case I : Sample with high concentration of analytes Y Y Y Low signal Case II : Sample with moderate concentration of analytes substrate Y Y Y Product (with colour) Moderate signal intensity Case III : Sample without analyte Y Y Y substrate Product (with colour) High signal intensity

12 Displacement affinity assay Primary antibodies were saturated with labelled analytes, followed by displacement of the labelled analytes by the sample analytes Signal reversely proportional to analyte concentration Suitable for small molecular weight analyte Lower sensitivity compare with direct assay

13 Sensitivity of affinity biosensors Direct affinity assay Imagine each candle representing an antibody and you are an optical transducer, you can easily detect if one candle light up (direct affinity assay) Competitive affinity assay You cannot easily detect if one candle is being off (competitive affinity assay)

14 Kinetics of affinity interactions Direct affinity interaction Competitive affinity interaction Kinetics of direct affinity interaction Kinetics of competitive affinity interaction Discussion: What are the associate constant and dissociation constant of affinity interaction?

15 Association and dissociation constant The association and dissociation constant is commonly used to describe the affinity between a ligand and a receptor (i.e. how tightly a ligand binds to a receptor). Ligand-receptor affinity are influenced by non-covalent intermolecular interactions between the ligand and receptor molecules such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces. [RL] Association constant = [R] [L] Dissociation constant = [R] [L] [RL] The larger the association constant, the higher the affinity between ligand and receptor The larger the dissociation constant, the smaller the affinity between ligand and receptor

16 Non-specific adsorption Supporting surface Ligand (analyte) can bound non-specifically to the supporting surface causing a false positive signal Reduce non-specific adsorption by blocking the supporting surface with (i) self-assembly monolayer or (ii) proteins e.g. bovine serum albumin (BSA).

17 Membrane-based lateral flow affinity devices (A) Flow through format (1980 S) Vertical flow Enzyme conjugates labels Multiple manual steps and reagents (B) Lateral flow format (1980 S till Today) Laterally flow across reactive zones, accumulation of labeled conjugates on test and control zone generates a measurable signal Colloidal gold conjugates labels One-step test concept (Whole blood assay)

18 Membrane-based affinity devices Membrane is inexpensive (~$6/m 2 ) which means individual cost per each paper based analytical devices is <$0.01. Membrane is thin, lightweight (~10mg/cm 2 ), and easy to store and transport. Membrane wicks fluids without the need of active pumping. Membrane is made of cellulose based materials which is compatible with biomaterials. Membrane can be disposed by incineration easily after use.

19 Classical lateral flow devices

20 Principle of lateral flow devices Optical transducer

21 Interpretation of lateral flow test Direct sandwich immunoassay (for large analyte) Presence of analytes in sample Absence of analytes in sample Flow direction Nano-gold biolabels bind with specific analyte forming a sandwich immuno-complex with the capture probes within the test zone Flow direction No immuno-complex formed within the test zone Analyte Capture probes Capture probe Test zone Test zone C T C T C T Case I: Sample contains analyte with concentration above the cut-off value Case II: Sample contains no analytes or with analyte concentration below cut-off value Case III: Invalid tests Red line show in the test zone indicate the presence of analytes

22 Luminex assay High throughput affinity assay on microbeads

23 Detection principle of Luminex assay

24 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

25 Amino linking of antibodies Early methods of immobilisation using the NH 2 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 NH 2 groups are used for the immobilisation because they are preferentially located in the recognition site. OR Inactive Active

26 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 Antibody Protein A Antibodies immobilization via Protein A

27 Receptors immobilization for improved sensitivity Protein A

28 Electrochemical affinity biosensors Analytical Chemistry, 82, , 2010.

29 QCM affinity biosensors QCM = Quartz crystal microbalance Analyst, 133, , 2008.

30 Magnetic affinity biosensors Journal of Immunological Methods, 338, 40-46, 2008.

31 Surface Plasmon Resonance (SPR)

32 Discussion and Exercise What is ELISA and how to perform a direct affinity assay? What is a ligand and a receptor? What is the meaning of dissociation constant and how the value (magnitude) of dissociation constant related to affinity interaction? What is used as label for lateral flow affinity device and which transducer used for detection of signal?

33 The end Please contact me if you have more questions. Dr. Wing Cheung Mak (Martin) Phone: Office: 2E:665