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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