GWC Technologies

The SPRimager®II is an array analysis system, designed for experiments where you want to monitor binding of one or more analytes to an array of probes.

The SPR 100 is a single-channel system designed for meticulous analysis of surface events (e.g. a probe-analyte interaction), simultaneously offering high sensitivity and very high dynamic range.

Should I use the SPRchip™ or SpotReady™ chip?

Use SpotReady™16 or SpotReady™25 chips when you are using the SPRimager®II and:

You need arrays with no more than 16 or 25 probes per chip
You don’t own robotic spotting equipment
You want to save the time and effort of setting up your robotic spotter, e.g. when you are developing biochip methods rather than running routine analyses
You need a large probe area for maximum detection sensitivity or for recovery of bound analyte for further analysis

Use SPRchip™ substrates when:

You are using the SPR100 wavelength scanning system; or
You are using the SPRimager®II and:
- You need arrays with more than 25 probes per chip
- You have a spotting robot and are running the same tests repeatedly

Can I use the same instrument and the same chips to detect Proteins vs Nucleic Acids vs ligands?
Yes. The SPR phenomenon is sensitive to changes in mass density on the surface, so anything that has mass is detectable. So you can perform completely different types of experiments on the same instrument, or you can analyze multiple types of molecules in the same experiment.

For examples of the types of experiments performed with GWC Technologies’ SPR systems, check out the applications page or contact your GWC Technologies representative.

How do I attach probes to SpotReady™ and SPRchip™ substrates?
There are many choices for probe attachment. Rather than force you to use a particular method for your experiments, GWC Technologies gives you complete freedom to choose your preferred method. And if you’re not sure what method to use, your GWC Technologies representative is available to help you, and can provide customers with detailed protocols.

Can I use the same surface chemistries with the SPRchip™ and SpotReady™?
Yes. Both chips have the same high quality, uniform gold surface.

Can I reuse SPRchip™ or SpotReady™ chips?
Yes. “Re-use” can have two distinct meanings:

1) Re-use the probe array.
This is readily feasible for reversible interactions—you may reverse the interaction either in situ by flowing fluid through the flow cell, or in a larger vessel ex situ, for example if you want to store the array for a while. In general:

DNA or RNA probe arrays used for nucleic acid hybridzation or protein binding can be re-used after washing. Use 8M urea for fastest washing, or simply water if time allows.

Antibody arrays may be reused if the antibody-antigen interaction is reversible under conditions that do not inactivate the antibody. For example, rinsing with polyols will remove antigen from polyol sensitive antibodies.

Metal ion-dependent binding is typically reversible by rinsing with buffer lacking the metal ion. For example, Subramanian et al (2006) demonstrated that ALG-2 binding to Annexin XI is readily reversed by removing Ca²⁺. Moreover, the Annexin XI array was reused successfully for ALG-2 detection after it was stored at 4° in buffer for 6 months.

RNA arrays used in AmpliFast™ tests are not reusable, because the RNA probes are degraded during detection.

Note that irreversible reactions will prevent re-use of the chips.

2) Re-use the chip from scratch by removing all surface chemistry.
GWC Technologies neither warrants chips beyond a single use, nor recommends re-use from scratch. Methods that effectively remove surface chemicals are dangerous to the user and may be deleterious to the high quality chip surface.

How sensitive is the SPRimager®II?
The SPRimager®II can detect a change in mass on the surface of <20 pg · mm^-2. Several factors influence how this translates to detection of biomolecular interactions:

the size of the analyte molecule—larger molecules are more readily detected;

the affinity of the interaction—higher affinity interactions are more readily detected;

the analyte concentration—higher concentrations are more readily detected;

the density of probe molecules—the more probe molecules per unit surface area, the more analyte molecules can bind, up to the limit of steric hindrance.

For more on sensitivity, refer to GWC’s Technical Note SPR Imaging Sensitivity. To find out if your specific experimental needs can be addressed, check out our applications page or contact your GWC Technologies representative.

How come DNA is detectable down to 1fM on the SPRimager®II? I didn’t think SPR was that sensitive
You can achieve high-sensitivity detection of specific DNA targets using GWC Technologies’ proprietary AmpliFast™ method of on-chip amplification. The label-free array-based method uses RNA oligo probes, which are degraded in the presence of RNAseH only when target DNA hybridizes to them. Target DNA is not degraded, and amplifies the signal by further hybridizing to remaining RNA probe molecules. Since the method provides real-time data, you can quantify both abundant and rare targets on the same chip.

For more details on AmpliFast™, please contact your GWC Technologies representative. For licensing opportunities, please contact .

Can I monitor protein-small molecule interactions using GWC Technologies’ SPR systems?
Yes.

For protein arrays, the smallest molecule detected on the SPRimager®II has a mass of 650 daltons.

For smaller molecules, you may instead make small molecule arrays, as demonstrated for example by Kanoh et al., 2006 and Smith et al., 2003. Then you can easily monitor protein binding to small molecules on the SPRimager®II

If you are studying just one protein, you can attach that protein to an SPRchip™ and readily monitor binding of small molecules using the SPR100 system available from Thermo.

Are data values obtained on the SPRimager®II from one experiment to another directly comparable?
Yes. A distinguishing feature of the SPRimager®II is the ability to convert SPR responses into changes in percent reflectivity (Δ%R), an absolute physical unit of measure. This means you don’t have to settle for arbitrary relative units (RUs), and can make direct comparisons between data sets acquired not only from different experiments but from different SPRimager®II instruments. It’s also a great way to make robust comparisons of different array fabrication methods.

Can I obtain dextran-coated chips from GWC Technologies?
No, all of our chips are dextran-free. GWC Technologies strongly recommends you avoid immobilization of probes in entangled polymers, especially when quantitative data are desired. Entangled polymers impede access of analyte to probe molecules, thereby compromising measured interaction properties.

What is a “difference image”?
A difference image is the result of digitally subtracting one image from another. Difference images are the primary data type for SPR imaging analysis. The process of creating the difference images is a key element in the robustness of SPR imaging measurements.

For more information on the SPR imaging method, please see GWC’s Technical Note, Introduction to SPR imaging.

I want to monitor binding of a protein to DNA, then measure binding of a second protein to the first one. Is this feasible?
Yes, you can do this type of analysis on both the SPRimager®II and SPR100 systems. In fact, you can monitor binding of several molecules sequentially on both instruments. And the SPR100 has such a large dynamic range you could monitor a dozen or more sequential binding events.

For advice on monitoring multiple sequential binding events, please contact your GWC Technologies representative.

How many samples can I spot on an SPRchip?
You have tremendous flexibility regarding the number and arrangement of probes on SPRchip™ substrates. 96-probe arrays, prepared using robotic spotting, are in frequent use. The smallest spot size we recommend for routine use is 50µm diameter, though spots as small as 10µm have been imaged on the SPRimager®II.

For more information on spatial issues please see GWC’s Technical Note, SPR imaging Spatial Resolution or contact your GWC Technologies representative.

Can I analyze molecular interactions from cell lysates using SPR detection?
The unique ability of the SPRimager® to obtain difference images coupled to the array format that allows negative controls to be exposed to the analyte at the same time, make this experiment feasible (e.g. Kyo et al., 2005). Be sure to incorporate positive and negative controls on your array so you can check the interaction is working and correct for nonspecific binding. Avoid reagents with a very high refractive index (such as glycerol).

Can I spot probes dissolved in organic solvents onto SpotReady™ chips?
While SpotReady™ chips are designed for aqueous solutions, they work well with probes dissolved in DMSO and DMF, but not for ethanol.

For additional information on using SpotReady™ chips, please contact your GWC Technologies representative.