Theory, Applications & Techniques. The BIACore 3000 Theory, Applications & Techniques.

Topics: How does it work? -Solid Phase ligand binding -Surface Plasmon Resonance What can it do? -Realtime Binding Kinetics. -Orphan Receptor Studies. What can you do? -Data Analysis -Some Examples so far...

Basic Principle A binding molecule is bound to the sensor surface.(eg a peptide) Another (the analyte) is passed over the surface and binds to it.

Study Methods Direct coupling of Ligand to Surface. Indirect, via a capture molecule (eg a specific IgG). Direct coupling of Ligand to Surface. Membrane anchoring, where the interacting ligand is on the surface of a captured liposome.

Sensor Chips: CM5 SA NTA HPA Pioneer Chips

Sensor Chip CM-5: Carboxymethylated dextran coated surface. Allows covalent coupling via -NH2, -SH, -CHO & -COOH groups:

Sensor Chip SA : Streptavidin coated dextran surface. Capture biotinylated DNA, proteins, lipids etc.

Bind His-tagged ligands to chelated nickel Sensor Chip NTA: Bind His-tagged ligands to chelated nickel

Anchor membrane-bound ligands on a hydrophobic surface Sensor Chip HPA: Anchor membrane-bound ligands on a hydrophobic surface

Pioneer Chips Pioneer Chip L1 (99-1000-05): Lipophilic Surface Facilitates the formation of lipid bilayers

Pioneer Chips: Pioneer Chip C1 (99-1000-04): Flat carboxymethylated surface. Useful for work with bulky components, such as cells and virus particles Pioneer Chip B1 (99-1000-02): Low degree of carboxylation Reduces non-specific binding of molecules which have a high positive charge, eg cell culture supernatants.

Pioneer Chips Pioneer Chip F1 (99-1000-03): Shortened dextran matrix Large analytes such as cells and virus particles Pioneer Chip J1 (99-1000-01): Gold surface Allows design of customized surface chemistry using self-assembled monolayers or other modifications

The Flow Cell F1 F2 F3 F4 F1 & 2 F3 & 4 F1 - 3 F1 - 4 Surface is divided into 4 channels, which can be used individually or in a number of combinations.

Microfluidic System Low reagents consumption Efficient mass transport Low dispersion Highly reproducible injections; CV typically less than 1% Wide range of contact times, 1 s - 12 h Sample recovery and fractionation

Measurement of Binding. Binding is measured as a change in the refractive index at the surface of the sensor. This is due to ‘Surface Plasmon Resonance’ (SPR). The change in refractive index is essentially the same for a given mass concentration change. (allows mass/concentration deductions to be made) Binding events are measured in real time. (allowing separate on and off rates to be measured.)

The Theory behind it. How? Binding is measured as a change in the refractive index at the surface of the sensor… How?

Total Internal Reflection At a certain angle of incidence, light entering a prism is totally internally reflected. (TIR). Although no photons exit the reflecting surface, their electric field extends ~1/4 wavelength beyond the surface.

Surface Plasmon Resonance If a thin gold film is placed on the reflecting surface, the photons can interact with free electrons in the gold surface. Under the right conditions, this causes the photons to be converted into plasmons and the light is no longer reflected.

Surface Plasmon Resonance This occurs when the incident light vector is equal to the surface plasmon vector….

Effect of binding on SPR. Plasmons create an electric field (evanescant) that extends into the medium surrounding the film. This is affected by changes in the medium (eg binding of analyte), and results in a change in the velocity of the plasmons. This change in velocity alters the incident light vector required for SPR and minimum reflection.

How does BIACore Measure this? Fixed wavelength light, in a fan-shaped form, is directed at the sensor surface and binding events are detected as changes in the particular angle where SPR creates extinction of light.

The Sensorgram The progress of an interaction is monitored as a sensorgram. Analyte binds to the surface-attached ligand during sample injection, resulting in an increase in signal. At the end of the injection, the sample is replaced by a continuous flow of buffer and the decrease in signal now reflects dissociation of interactant from the surface-bound complex. A response of 1000 RU corresponds to a change in surface concentration of 1 ng/mm2.

Binding Analysis How Much? Active Concentration. How Fast? Kinetics. How Strong? Affinity. How Specific? Specificity.

Concentration. Signal proportional to mass. Same specific response for different proteins.

Binding Kinetics Real-time ‘association’ and ‘dissociation’ rates. Analysis of bivalent, multimeric and heterogenous analytes. Analytes from around 340 Da to whole cells.

Binding Kinetics dissociation association ka kd

Using it... Stage One Choose ligand. Choose chip/immobilisation method.

Sensor Chips: CM5 SA NTA HPA Pioneer Chips

Using it... Stage One Choose ligand. Choose chip/immobilisation method. Choose immobilisation levels. Immobilise ligand.

Immobilisation Levels. Low High Specificity Concentration Affinity Kinetics LMW binding

Using it... Stage Two Choose analyte. Choose regeneration method. Choose type of analysis required.

BIACore Software. BIAControl. BIAEvaluation. BIASimulation. Controls the BIACore and records the sensorgram. BIAEvaluation. Kinetic analysis of sensorgrams. BIASimulation. Allows simulation of various binding situations..

Types of Analysis Surface Preparation ‘wizard’ Kinetics analysis ‘wizard’ Manual operation Custom methods (eg. MICRORECOVER)

Surface Preparation Wizard Immobilising ligand using defined conditions or to a preset target level. Preparing reference surfaces for inline reference subtraction. Testing analyte binding capacity and regenration conditions

Kinetic Analysis Wizard Analysis using captured ligand Determination of kinetic parameters from analyte concentration series Control experiments for mass transfer, linked reactions and kinetic heterogeneity.

Manual Operation Manual setting of : detection mode, flow path flow rates contact times injection volumes…

Custom Methods Allows complete control of automated procedures. eg MICRORECOVER method for recovery of bound analyte in small (µl) volumes.

BIACore Software. BIAControl. BIAEvaluation. BIASimulation. Controls the BIACore and records the sensorgram. BIAEvaluation. Kinetic analysis of sensorgrams. BIASimulation. Allows simulation of various binding situations..

All in a days work! 9am: Immobilisation of ligand to the sensor chip 10am: Set up and start analysis wizard (approx 3 hours per analyte) 2pm: Use of BIAEvaluation to analyse results 4pm: Printing of results Last post: Submission to Nature The progress of an interaction is monitored as a sensorgram. Analyte binds to the surface-attached ligand during sample injection, resulting in an increase in signal. At the end of the injection, the sample is replaced by a continuous flow of buffer and the decrease in signal now reflects dissociation of interactant from the surface-bound complex. A response of 1000 RU corresponds to a change in surface concentration of 1 ng/mm2.

Further information. Further info and examples can be found under “BIACore” on the “Facilities” page, in the “General Information” section of the AMS website: http://www.ams.rdg.ac.uk/info/facilities.html The progress of an interaction is monitored as a sensorgram. Analyte binds to the surface-attached ligand during sample injection, resulting in an increase in signal. At the end of the injection, the sample is replaced by a continuous flow of buffer and the decrease in signal now reflects dissociation of interactant from the surface-bound complex. A response of 1000 RU corresponds to a change in surface concentration of 1 ng/mm2.