Surface Plasmon Resonance (SPR) -BIAcore T200

 
 

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Instrument:

The BIAcore T200 instrument is located in Swann Rm. 3.10. Please follow the link for information regarding the Booking and rules for use of the BIAcore T200 instrument:


Download technical information on the T200 system.

For more information on SPR technology and BIAcore's products: www.biacore.com

Please click here for individual platform/resource costings.

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What is SPR?

The underlying physics of the principles of SPR are complicated. However, a working knowledge of the technique doesn't require a detailed understanding of the theory. Basically, SPR-based instruments use an optical method to measure the refractive index near (within ~ 300 nm) the surface of a sensor. In BIAcore instruments this surface is one side of a micro-fluidic flow cell (~ 20 - 60 nL). Through this flow cell an aqueous solution (running buffer) is passed under a continuous flow rate (1 - 100 µL/min). To allow the detection of an interaction one molecule (termed by BIAcore as the ligand) is immobilized onto the sensor surface. Its binding partner (termed by BIAcore as the analyte) is then injected in aqueous solution (ideally with the same components and composition as the running buffer) through the flow cell, under continuous flow. As the analyte binds to the ligand the accumulation of protein on the sensor surface causes an increase in refractive index. This refractive index change is measured in real time (sampling in a kinetic analysis experiment is taken every 0.1 s), and the result plotted as response units (RU) versus time (termed a sensorgram). Importantly, a response (background response) will also be generated if there is a  difference in the refractive indices of the running and sample buffers. This background response must be subtracted from the sensorgram to obtain the actual binding response. The background response is recorded by injecting the analyte through a control or  reference flow cell, which has no ligand or an irrelevant ligand  immobilized to the sensor surface. The real time measurement of  association and dissociation of a binding interaction allows for the  calculation of association and dissociation rate constants and the corresponding affinity constants. One RU represents the binding of 1 pg of protein per square mm. More than 50 pg per square mm of analyte binding is needed in practice to generate good  reproducible responses, and is it practically difficult to immobilize a sufficiently high density of ligand directly onto a sensor surface  to achieve this level of analyte binding. To get round this technical difficulty, BIAcore sensor chips have a 100 - 200 nm thick carboxymethylated dextran matrix attached. This adds a third dimension to the surface, resulting in the ability to generate much  higher levels of ligand immobilization. In addition, the  carboxymethylated dextran matrix is very amenable to modification for  covalent immobilization reactions by a variety of chemistries. E.g. primary amine, SH, aldehyde. However, having very high levels of ligand immobilised on the surface has two important implications for the technique. Such high ligand density may result in the rate at which the surface binds the analyte exceeding the rate at which the analyte can be delivered to the surface (this latter consideration is referred to as mass transport). If this condition arises, mass transport becomes the rate-limiting step, and as a consequence, the measured apparent on-rate will be slower than the true on-rate. A related problem is that, following dissociation of the analyte, it can rebind to the unoccupied ligand before diffusing out of the matrix and being washed from the flow cell.  Consequently, the measured apparent off-rate is slower than the actual off-rate. Although the dextran matrix may exaggerate these kinetic artifacts  (mass transport limitations and re-binding) they can affect all  surface-binding techniques.

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More detail ...

The SPR optical unit consists of a source for a light beam that passes through a prism and strikes the surface of a flow cell at an angle, such that the beam is totally reflected. Under these conditions, an electromagnetic component of the beam, the evanescent wave, propagates into the aqueous layer and can interact with mobile electrons in the gold film at the surface of the glass. At a particular wavelength and incident angle, a surface plasmon wave of excited electrons (the plasmon resonance) is produced at the gold layer and is detected as a reduced intensity of the reflected light beam. In Biacore instruments, monochromatic light in the shape of a wedge (a broad distribution of incident angles) is used.


Each angle of the reflected beam strikes the instrument's detector at a different point and, thus, the detector continuously records the position of reduced light intensity and calculates the SPR angle from that figure. The optical device has no moving parts and the fixed geometry enhances stability and allows binding to be monitored in real time. The SPR angle is sensitive to the composition of the layer at the surface of the gold. A baseline SPR angle is first determined by washing buffer over the surface with a fixed amount of ligand attached. To this flow of buffer, some analyte is then added. The binding of analyte to immobilized ligand causes an increase in the refractive index at the surface, thereby changing the SPR angle because it is directly proportional to the amount of bound analyte. No labeling of molecules is required in the SPR detection method, and the binding of probes with molecular weights ≥ 200 Da. can be detected quite accurately (see above). The SPR angle change is reported as resonance units (RU), where 1000 RU correspond to an angle change of ~ 0.1º. For most proteins, binding of ~ 1 ng per square mm of protein at the dextran surface is required to cause a signal change of 1000 RU. The exact relation between RU and ng of material bound will vary with the refractive index of the analyte. If the added molecule does not bind to a target or receptor, the SPR angle change in the sample and reference flow cells will be the same, and, after subtraction, will give a zero net RU response that indicates no binding occurred. Only bound protein generates a positive SPR signal and that signal, recorded over time, produces a sensorgram.

Surface generation and results: an example.

 

Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Lab 3.10

SPR - BIAcore T200

contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Snr. Protein Technologist:

Dr. Liz Blackburn

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: E.A.Blackburn@ed.ac.uk


Snr. Protein Technologist:

Dr. Matt Nowicki

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: matthew.nowicki@ed.ac.uk



Facilities:


CTCB-Home

Protein Production

Biophysical Characterisation

In Silico Screening

Training


Links:


On-line Booking

Core Column Library


Access Charges/Costs:


Access Charges/Costs