Post on 30-May-2018
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Biacore™ Basics
Introduction to SPR Analysis
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Objectives
During this session you will get introduced to
• Biacore data
– What type of analyses can be performed
– The sensorgram
• Biacore technology
– Surface Plasmon Resonance (SPR)
– Microfluidics
– Sensor Chips
• General steps in a Biacore assay
– Immobilization
– Sample injection
– Regeneration
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Biacore systems monitor molecular binding events in real-time
Real-time plot of binding response over time (sensorgram)
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What are ligand and analyte?
The ligand is the interaction partner attached to the surface
Analyte
Ligand
The analyte is the interaction partner
that passes in solution over the surface
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Data output from label-free interaction analysis
baseline
buffer
Time
Response (RU)
Sensorgram
sample
association
buffer
dissociation
report point
bindingresponse
curve shape revealskinetics of interaction
Yes/no binding?
Specificity of binding?
How strong?
How fast?
How much?
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Comprehensive information from one system
Analyze molecular interactions in real time and obtain a wide range of
critical, binding-related data:
DetectDetect
Yes/No
IdentifyIdentify
Specificity
Binding partners
CharacterizeCharacterize
Affinity
Kinetics
Concentration
Thermodynamics
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Analysis of a wide range of biomolecularinteractions
• Proteins
• Nucleic acids
• Lipids & membrane-associated molecules
• Carbohydrates
• LMW compounds (>200 Da)
• Whole cells
• Viruses/bacteria
CD4/gp120
LMW compound
mAb
Detergent-solubilizedGPCRs
natural chemokine
Ab captured onsensor surface
CD4/gp120
LMW compound
mAb
Detergent-solubilizedGPCRs
natural chemokine
Ab captured onsensor surface
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Biacore™ data in almost 10 000 scientific publications
Basic and applied research in the fields of
• Cancer
• Neurobiology
• Immunology
• Infectious diseases
• Functional proteomics
• Cell signaling
• Vaccines
• Selection and characterization of binding reagents
• Drug discovery
and many more….
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Cornerstones of the system
Microfluidic system
SPR detection
system
Sensor chip
User-friendly software
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SPR detection
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Total Internal Reflection & SPR
High refractive index medium: Prism
Low refractive index medium: water
Gold layer
Incident Light Total Internal Reflected light (TIR)
Critical angle for TIR
evanescent field
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SPR SensingMeasuring at the Surface
Distance [nm]
Sen
sitivity
Glass
Me
tal fi
lm
Dextran + immobilized protein
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SPR detection
• SPR is a refractive index sensor
• Measurements are dependent on surface concentration & temperature
• SPR response is a measure of changes in the resonance angle
• As a rough approximation, a response of 1 RU is equivalent to change in a
surface concentration of about 1 pg/mm2 (for proteins on Sensor Chip CM5)
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MicrofluidicsLiquid handling
• Miniaturized system
• Low volume of reagents
• Integrated and automated liquid handling
IFC
Flow cells
(expanded view)
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Biacore technology: MicrofluidicsFlow cells
• The number of flow cells and the type of flow varies between different Biacore systems
Serial flow
(4 flow cells)
-Biacore 2000
-Biacore 3000-Biacore T100
Parallel flow
(4 flow cells)
-Biacore 1000
-Biacore C-Biacore Q
Serial flow(2 flow cells)
-Biacore X
-Biacore X100-Biacore J
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Microfluidics – Flow cells
4 flow cells – single, pair wise and serial run
Flow cells optimized for use in pairs (FC1+FC2, FC3+FC4)
FC 1
FC 2
FC 3
FC 4
IN
OUTOpen valve
Closed valve
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Buffer Selector
Buffer selector valve
• Assay development
• Study binding in different environments (e.g. pH, salt)
• Controlled from the software
• Up to 4 different buffers can be used
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Integrated buffer degasser
Integrated buffer degasser
• Eliminates precipitation of air in the flow system
• Buffers do not need to be degassed externally
Improves robustness when studying interactions at temperatures > 25 °C
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Liquid handling system
Waste
Syringepumps
Peristalticpump
WaterBuffer
Buffer selectorvalve
A B C D
IFC
Vacuumdegasser
S2
To waste
S1
Needle
Buffer
degasser
Buffer selector
valve
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The sensor chip
Dextran matrix
Linker layer
Gold
Glass support
Carboxyl group
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Dextran matrix
• Hydrophilic
• Flexible
• Resembles a 2% aqueous dextran solution environment
• Low non-specific binding
• High binding capacity
• Easy to activate and use for covalent coupling
• Withstands extensive regeneration
• Present on all sensor chips except Sensor Chip C1, Sensor Chip HPA and Sensor Chip Au
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Sensor Chip CM5
For covalent attachment of ligand
or capturing molecule using
• Amine coupling
• Ligand thiol coupling
• Surface thiol coupling
• Maleimide coupling
• Aldehyde coupling CM5• The most versatile chip
• Carboxylated dextran matrix
• Low non-specific binding
• High binding capacity
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A wide range of sensor chips
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General Steps in a Biacore Assay
Surface preparation
Sample injection
Regeneration
Evaluation
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Surface Preparation - Immobilization
• What is immobilization?
– Coupling of ligand or capture molecule to the sensor surface
• Direct immobilization
– Covalent coupling of ligand to surface
• Capture approach
– Covalent coupling of capture molecule
– Capture of ligand during each cycle via a high-affinity interaction
Analyte
Ligand
Analyte
Ligand
Capturing molecule
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Sample injection
• The sample is injected over the surface with a constant flow and
concentration
• Analyte in the sample binds to the immobilized ligand on the surface, the
mass on the surface changes and a response is recorded
• After sample injection, buffer flows over the surface to allow monitoring of
the dissociation of analyte from ligand
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Regeneration
• Removes bound analyte completely from the surface
• The activity of the surface must remain unaffected
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The Sensorgram
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Summary
• Biacore technology
– SPR
– Sensor Chip Surfaces
– Microfluidics
• Important Biacore Assay Steps
– Immobilization
– Sample injection
– Regeneration
• Biacore data
– Includes specificity, kinetics, affinity
and concentration
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Allows flexible assay design
and analysis of a wide range
of molecular interactions
Results in detailed
characterization and analysis
of biomolecular interactions
Gives information-rich real
time data