The BIACore 3000 Theory, Applications & Techniques.

The BIAThe BIACore Core 30003000

Theory, Applications & Techniques.Theory, Applications & Techniques.

Topics:Topics:

• How does it work?How does it work?-S-Solid Phase ligand bindingolid Phase ligand binding

--Surface Plasmon ResonanceSurface Plasmon Resonance

What can it do?What can it do?--Realtime Binding Kinetics.Realtime Binding Kinetics.

-Orphan Receptor Studies.-Orphan Receptor Studies.

• What can you do?What can you do?--Data AnalysisData Analysis

--Some Examples so far...Some Examples so far...

Basic PrincipleBasic Principle

• A binding molecule is bound to the sensor surface.(eg A binding molecule is bound to the sensor surface.(eg a peptide)a peptide)

• Another Another (the analyte)(the analyte) is passed over the surface and is passed over the surface and binds to it.binds to it.

Study MethodsStudy Methods

– Direct coupling of Ligand to Surface.Direct coupling of Ligand to Surface.

– Membrane anchoring, where the interacting ligand is on the surface of a Membrane anchoring, where the interacting ligand is on the surface of a captured liposome.captured liposome.

– Indirect, via a capture molecule (eg a specific IgG).Indirect, via a capture molecule (eg a specific IgG).

CM5CM5

SASA

NTANTA

HPAHPA

Pioneer ChipsPioneer Chips

Sensor Chips:Sensor Chips:

Allows covalent coupling via -NHAllows covalent coupling via -NH22, -SH, -CHO & -COOH groups:, -SH, -CHO & -COOH groups:

Sensor Chip CM-5:Sensor Chip CM-5: Carboxymethylated dextran coated surfaceCarboxymethylated dextran coated surface..

Sensor Chip SA :Sensor Chip SA : Streptavidin coated dextran surface.Streptavidin coated dextran surface.

Capture biotinylated DNA, proteins, lipids etc.Capture biotinylated DNA, proteins, lipids etc.

Sensor Chip NTA: Sensor Chip NTA:

Bind His-tagged ligands to chelated nickelBind His-tagged ligands to chelated nickel

Sensor Chip HPA:Sensor Chip HPA:

Anchor membrane-bound ligands on a hydrophobic surfaceAnchor membrane-bound ligands on a hydrophobic surface

Pioneer ChipsPioneer Chips

•Pioneer Chip L1 (99-1000-05): Pioneer Chip L1 (99-1000-05): Lipophilic SurfaceLipophilic Surface

Facilitates the formation of lipid bilayersFacilitates the formation of lipid bilayers

Pioneer Chips:Pioneer Chips:

•Pioneer Chip B1 (99-1000-02): Pioneer Chip B1 (99-1000-02): Low degree of carboxylation Low degree of carboxylation

Reduces non-specific binding of molecules which have a high Reduces non-specific binding of molecules which have a high positive charge, eg cell culture supernatants.positive charge, eg cell culture supernatants.

•Pioneer Chip C1 (99-1000-04): Pioneer Chip C1 (99-1000-04): Flat carboxymethylated surface.Flat carboxymethylated surface.

Useful for work with bulky components, such as cells and Useful for work with bulky components, such as cells and virus particlesvirus particles

Pioneer ChipsPioneer Chips

•Pioneer Chip F1 (99-1000-03): Pioneer Chip F1 (99-1000-03): Shortened dextran matrix Shortened dextran matrix

Large analytes such as cells and virus particlesLarge analytes such as cells and virus particles

•Pioneer Chip J1 (99-1000-01): Pioneer Chip J1 (99-1000-01): Gold surfaceGold surface

Allows design of customized surface chemistry using self-Allows design of customized surface chemistry using self-assembled monolayers or other modificationsassembled monolayers or other modifications

The Flow CellThe Flow Cell

F1F1

F2F2

F3F3

F4F4

F1 & 2F1 & 2

F3 & 4F3 & 4

F1 - 3F1 - 3

F1 - 4F1 - 4

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

Microfluidic SystemMicrofluidic System

• Low reagents consumptionLow reagents consumption

• Efficient mass transportEfficient mass transport

• Low dispersionLow dispersion

• Highly reproducible injections; CV typically less than 1%Highly reproducible injections; CV typically less than 1%

• Wide range of contact times, 1 s - 12 hWide range of contact times, 1 s - 12 h

• Sample recovery and fractionationSample recovery and fractionation

Measurement of Binding.Measurement of Binding.

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

• This is due to ‘Surface Plasmon Resonance’ (SPR).This is due to ‘Surface Plasmon Resonance’ (SPR).

• The change in refractive index is essentially the same for a The change in refractive index is essentially the same for a given mass concentration change. given mass concentration change. (allows mass/concentration (allows mass/concentration deductions to be made)deductions to be made)

• Binding events are measured in real time. Binding events are measured in real time. (allowing separate on and off rates to be measured.)(allowing separate on and off rates to be measured.)

The Theory behind it.The Theory behind it.

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

How?How?

•At a certain angle of incidence, light entering a prism is At a certain angle of incidence, light entering a prism is totally internally reflected. (TIR).totally internally reflected. (TIR).

Total Internal ReflectionTotal Internal Reflection

•Although no photons exit the reflecting surface, their Although no photons exit the reflecting surface, their electric field extends ~1/4 wavelength beyond the surface.electric field extends ~1/4 wavelength beyond the surface.

Incident angle ( θ) < critical angle for TIR REFRACTION θ θIncident angle ( θ) > critical angle for TIR TOTAL INTERNAL REFLECTION

Surface Plasmon ResonanceSurface Plasmon Resonance

•If a thin gold film is placed on the reflecting surface, the If a thin gold film is placed on the reflecting surface, the photons can interact with free electrons in the gold surface.photons can interact with free electrons in the gold surface.

λλ14Free Electrons

•Under the right conditions, this causes the photons to be Under the right conditions, this causes the photons to be converted into plasmons and the light is no longer reflected.converted into plasmons and the light is no longer reflected.

Plasmons

Surface Plasmon ResonanceSurface Plasmon Resonance

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

Incident light vector ≠ Surface plasmon vectorMismatch: No resonance Full Reflection

θ1••• Gold surfaceθ1Angle Incident light vector = Surface plasmon vectorMatch: Resonance Minimum Reflection

θ2••• Gold surfaceθ2Angle

•Plasmons create an electric field Plasmons create an electric field (evanescant)(evanescant) that extends into that extends into the medium surrounding the film.the medium surrounding the film.

•This is affected by changes in the medium (eg binding of This is affected by changes in the medium (eg binding of analyte), and results in a change in the velocity of the plasmons.analyte), and results in a change in the velocity of the plasmons.

•This change in velocity alters the incident light vector required This change in velocity alters the incident light vector required for SPR and minimum reflection.for SPR and minimum reflection.

Effect of binding on SPR.Effect of binding on SPR.

How does BIACore Measure this? How does BIACore Measure this?

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

The SensorgramThe Sensorgram

Binding AnalysisBinding Analysis

• How Much?How Much? Active ConcentrationActive Concentration..

Kinetics.Kinetics.

Affinity.Affinity.

Specificity.Specificity.

• How Fast?How Fast?

• How Strong?How Strong?

• How Specific?How Specific?

Concentration.Concentration.

• Signal proportional to Signal proportional to mass.mass.

• Same specific response Same specific response for different proteins.for different proteins.

Binding KineticsBinding Kinetics

• Real-timeReal-time ‘association’ and ‘dissociation’ rates. ‘association’ and ‘dissociation’ rates.

• Analysis of bivalent, multimeric and heterogenous analytes.Analysis of bivalent, multimeric and heterogenous analytes.

• Analytes from around 340 Da to whole cells.Analytes from around 340 Da to whole cells.

Binding KineticsBinding Kinetics

association

ka

dissociation

kd

Using it...Using it...

• Stage OneStage One

– Choose ligand.Choose ligand.

– Choose chip/immobilisation method.Choose chip/immobilisation method.

CM5CM5

SASA

NTANTA

HPAHPA

Pioneer ChipsPioneer Chips

Sensor Chips:Sensor Chips:

Using it...Using it...

• Stage OneStage One

– Choose ligand.Choose ligand.

– Choose chip/immobilisation method.Choose chip/immobilisation method.

– Choose immobilisation levels.Choose immobilisation levels.

– Immobilise ligand.Immobilise ligand.

Immobilisation Levels.Immobilisation Levels.

LowLow HighHigh

– ConcentrationConcentration

– AffinityAffinity

– KineticsKinetics

– LMW bindingLMW binding

– SpecificitySpecificity

Using it...Using it...

• Stage TwoStage Two

– Choose Choose analyte.analyte.

– Choose regeneration method.Choose regeneration method.

– Choose type of analysis required.Choose type of analysis required.

BIACore Software.BIACore Software.

• BIABIAControlControl..– Controls the BIACore and records the sensorgram.Controls the BIACore and records the sensorgram.

• BIABIAEvaluationEvaluation..– Kinetic analysis of sensorgrams.Kinetic analysis of sensorgrams.

• BIABIASimulationSimulation..– Allows simulation of various binding situations..Allows simulation of various binding situations..

Types of AnalysisTypes of Analysis

• Surface Preparation ‘wizard’Surface Preparation ‘wizard’

• Kinetics analysis ‘wizard’Kinetics analysis ‘wizard’

• Manual operationManual operation

• Custom methodsCustom methods

– (eg. M(eg. MICRORECOVERICRORECOVER))

Surface Preparation WizardSurface Preparation Wizard

• Immobilising ligand using defined conditions or to a Immobilising ligand using defined conditions or to a preset target level.preset target level.

• Preparing reference surfaces for inline reference Preparing reference surfaces for inline reference subtraction.subtraction.

• Testing analyte binding capacity and regenration Testing analyte binding capacity and regenration conditionsconditions

Kinetic Analysis WizardKinetic Analysis Wizard

• Analysis using captured ligandAnalysis using captured ligand

• Determination of kinetic parameters from analyte Determination of kinetic parameters from analyte concentration seriesconcentration series

• Control experiments for mass transfer, linked reactions Control experiments for mass transfer, linked reactions and kinetic heterogeneity.and kinetic heterogeneity.

Manual OperationManual Operation

• Manual setting of :Manual setting of :

– detection mode, detection mode,

– flow pathflow path

– flow ratesflow rates

– contact timescontact times

– injection volumes…injection volumes…

Custom MethodsCustom Methods

• Allows complete control of automated procedures.Allows complete control of automated procedures.

• eg Meg MICRORECOVERICRORECOVER method method

– for recovery of bound analyte in small (µl) volumes.for recovery of bound analyte in small (µl) volumes.

BIACore Software.BIACore Software.

• BIABIAControlControl..– Controls the BIACore and records the sensorgram.Controls the BIACore and records the sensorgram.

• BIABIAEvaluationEvaluation..– Kinetic analysis of sensorgrams.Kinetic analysis of sensorgrams.

• BIABIASimulationSimulation..– Allows simulation of various binding situations..Allows simulation of various binding situations..

All in a days work!All in a days work!

•9am:9am: Immobilisation of ligand to the sensor chipImmobilisation of ligand to the sensor chip

•10am:10am: Set up and start analysis wizard Set up and start analysis wizard

(approx 3 hours per analyte)(approx 3 hours per analyte)

•2pm:2pm: Use of BIAEvaluation to analyse resultsUse of BIAEvaluation to analyse results

•4pm:4pm: Printing of resultsPrinting of results

•Last post:Last post: Submission to NatureSubmission to Nature

Further information.Further information.

Further info and examples can be foundFurther info and examples can be found

underunder “BIACore” “BIACore”

on theon the “Facilities” “Facilities” page, page,

in thein the “General Information” “General Information”

section of the AMS website:section of the AMS website:

http://www.ams.rdg.ac.uk/info/facilities.htmlhttp://www.ams.rdg.ac.uk/info/facilities.html