The BIACore 3000

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The BIACore 3000

• Theory, Applications Techniques.

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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...

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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.

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Study Methods
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Sensor Chips
CM5 SA NTA HPA Pioneer Chips
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Sensor Chip CM-5 Carboxymethylated dextran
coated surface.
Allows covalent coupling via -NH2, -SH, -CHO
-COOH groups
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Sensor Chip SA Streptavidin coated dextran
surface.
Capture biotinylated DNA, proteins, lipids etc.
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Sensor Chip NTA
Bind His-tagged ligands to chelated nickel
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Sensor Chip HPA
Anchor membrane-bound ligands on a hydrophobic
surface
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Pioneer Chips

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

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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.

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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

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The Flow Cell
Surface is divided into 4 channels, which can be
used individually or in a number of combinations.
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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

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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.)

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The Theory behind it.

• Binding is measured as a change in the refractive
  index at the surface of the sensor

How?
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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.

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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.

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Surface Plasmon Resonance

• This occurs when the incident light vector is
  equal to the surface plasmon vector.

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• 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.

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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.

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The Sensorgram
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Binding Analysis

• How Much?

Active Concentration.
Kinetics.

• How Fast?

Affinity.

• How Strong?

Specificity.

• How Specific?

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Concentration.

• Signal proportional to mass.

• Same specific response for different proteins.

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Binding Kinetics

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

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Binding Kinetics
dissociation
association
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Using it...

• Stage One
• Choose ligand.
• Choose chip/immobilisation method.

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Sensor Chips
CM5 SA NTA HPA Pioneer Chips
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Using it...

• Stage One
• Choose ligand.
• Choose chip/immobilisation method.

• Choose immobilisation levels.
• Immobilise ligand.

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Immobilisation Levels.
Low
High
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Using it...

• Stage Two
• Choose analyte.
• Choose regeneration method.
• Choose type of analysis required.

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BIACore Software.

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

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Types of Analysis

• Surface Preparation wizard
• Kinetics analysis wizard
• Manual operation
• Custom methods
• (eg. MICRORECOVER)

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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

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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.

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Manual Operation

• Manual setting of
• detection mode,
• flow path
• flow rates
• contact times
• injection volumes

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Custom Methods

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

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BIACore Software.

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

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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

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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