BiaCore(BIA)Technology Real-time BIA is a technology developed by Pharmacia Biosensor AB for monitoring biomolecular interaction in real time, using a non-invasive optical detection principle based on surface plasmon resonance (SPR) BIAapplication

1
BiaCore(BIA)Technology Real-time BIA is a
technology developed by Pharmacia Biosensor AB
for monitoring biomolecular interaction in real
time, using a non-invasive optical detection
principle based on surface plasmon resonance
(SPR)BIAapplication Handbook, (Pharmacia
Biosencor AB, Uppsala, Sweden, 1998).

2
Surface Plasmon Resonance (SPR)
3
SPR Principle
4
SPR Biosensor
SPR-based biosensors. The sensor chip provides a
gold-coated surface through which minute changes
in biomolecule concentration, due to molecular
binding, are detected in real time. These changes
lead to changes in the reflected light that are
captured by the optical detection unit and
converted to a resonance signal. (Courtesy of
Biacore International AB.)
5
SPR Biosensors

• Surface plasmon resonance, or SPR, is a
  biophysical technique used to measure the binding
  interactions of very small amounts of a target
  protein. A ligand is immobilized on a special
  chip and a solution of the target molecule flows
  across the chip. The progress of binding is then
  measured by optical instruments.
  en.wikipedia.org/wiki/Surface_plasmon_resonance

6
PTC
Title Tech.Sight. Analyzing biomolecular
interactions. By Wilson WD, Science, 1095-9203,
2002 Mar 15, Vol. 295, Issue 5562

• target molecule (T) with a specific binding site
  (such as a particular region in a protein
  tertiary structure or a specific sequence of DNA)
  and a probe molecule (P) that can bind to that
  site. The simplest binding model corresponds to
  P T ? C, where C is the resulting complex

7
SPR Principle
8
BiaCore Technology

• 
  http//www.biacore.com/lifesciences/index.html
• 1 Carboxymethyl-dextran is linked to the gold to
  give the interaction layer
• 2 The ligand or probe molecules (T) must be
  linked to this layer to create the biospecific
  recognition surface
• 3 The target molecules flow through the surface
  and interact with the Ligand or Probe (P) to form
  the bindind complex (C)
• 4 SPR responds to changes in refractive index
  (mass change)

9
Real-time BIA Procedure

• Preparation of the biospecific surface
• Measurement of kinetic constants
• Measurement of analyte concentration
• Determination of binding patterns
• Regeneration of the surface

10
Preparation of the biospecific surface

• Chemistry of the coupling procedure
• 
  BIAapplication Handbook, (Pharmacia Biosencor AB,
  Uppsala, Sweden, 1998).

11
Preparation of the biospecific surface

• Recommended coupling chemistries for various
  ligand types and functional groups
• BIAapplication Handbook, (Pharmacia Biosencor AB,
  Uppsala, Sweden, 1998).

12
Measurement of kinetic constants

• Softwares
• BIAEvaluation Software (BiaCore)
• Scrubber (U. of Utah)
• Kinetics rates of reaction (ka)
• The kinetics of an interaction,
  i.e. the rates of complex formation (ka) and
  dissociation (kd), can be determined from the
  information in a sensorgram. Biacore
  evaluation software generates the values of ka
  and kd by fitting the data to interaction models.
• Affinity the strength of binding (Kd, Ka)
• The affinity of an interaction is
  determined from the level of binding at
  equilibrium (seen as a constant signal) as a
  function of sample concentration.
• Concentration (C)
• Concentration is determined by
  monitoring the interaction of a molecule with a
  prepared sensor surface in the presence of a
  target molecule in solution (solution inhibition)
  or excess analyte (surface competition).
  Concentrations are calculated by interpolation of
  the binding responses on a calibration curve.
• Interaction Pattern
• Complex formation can be monitored
  as each component is incorporated into a
  multimolecular complex. For example
• http//www.biacore.com/lifesciences/technology/int
  roduction/data_interaction/index.html

13
Measurement of kinetic constants

• 
  BIAapplication Handbook, (Pharmacia Biosencor AB,
  Uppsala, Sweden, 1998).

14
Regeneration of the Surface

• Reuse the chip surface
• Ligand stay intact
• Not cause any irreversible damage to the ligand
• Low pH buffer such as 10mM Glysin (pH 1.5 to 2.5)

15
Application

• Protein - Protein
• Protein - DNA
• Protein - RNA
• Protein - Carbohydrate
• Protein - Lipid
• Enzyme vs DNA
• Enzyme vs Insecticides
• Virus - Protein
• Platelet - Protein
• Small molecule - Protein