Frequently Asked Questions
Here's what you've been wondering about the WAVEsystem
What is the smallest analyte the Creoptix WAVEsystem can measure?
There is not a fixed molecular weight (MW) limit for the analyte, as a lot depends on the MW ratio between the ligand and the analyte, together with the stoichiometry and the “cleanliness” of the system. We routinely measure methyl sulfonamide (90 Da) and we have also measured nickel ions (58 Da) as analytes, but the signal level with any analyte depends on how many binding sites are (or can be) available on the surface (MW ratio plus stoichiometry), while the lower limit of usable signal for kinetics depends on the noise level (cleanliness).
The response in the Creoptix WAVEsystem is – as a rule of thumb – proportional to the MW of the measured molecule as well as to the number of molecules bound to the sensor. The high sensitivity of the Creoptix WAVEsystem allows for measuring robust sensorgrams at very low responses (< 1 pg/mm2), however, the maximal response (Rmax) of the system will depend on the number of immobilized ligand molecules / epitopes as well as the kinetics of the interaction. Generally, for ligand-analyte pairs with a large difference in MW (large MW ratio), the maximal response is more limited, as large ligands at the maximal density (saturation of the matrix) have fewer molecules (binding sites) available for interaction. The Creoptix WAVEsystem can provide robust binding responses for interactions with a MW ratio of up to 1:1000 (i.e. a 500 Da small molecular analyte binding to a 500 kDa protein).
How do you know that 1 pg/mm2 on the Creoptix WAVEsystem equates to 1 RU in surface Plasmon Resonance (SPR) devices?
1 pg/mm2 of proteins is considered equivalent to 1 RU in SPR (Stenberg et al, J. Colloid Interface Sci., 143 (1991) 513-526). Mathematically, the refractive index changes on the surface that give 1 pg/mm2 signal on the Creoptix WAVEsystem are equivalent to 1 RU on Cytiva’s Biacore.
What are the weakest and strongest affinities the Creoptix WAVEsystem can measure?
In a well optimized assay, the Creoptix WAVEsystem can measure very slow and very fast interaction rates, with the on-rate (kon) ranging from 103 to 106 M-1 s-1 and the off-rate (koff) ranging from 10-6 to 10 s-1. This allows for assessment of a very broad affinity range, from low pico- to sub-millimolar.
In terms of practical considerations for experimental design, the limit for measurable high-affinity depends on the noise of the tested system, while the limit for measurable low-affinity is set by the solubility of the analyte.
How is GCI different from Bio-Layer Interferometry (BLI)?
Although both GCI and BLI work by using interference to measure refractive index changes on a thin layer above the surface of the sensor, they are two completely different technologies. GCI, the technology used in the Creoptix WAVEsystem, measures the effect of refractive index changes on an evanescent wave generated by the light passing through the waveguide in the sensor. These refractive index changes affect the phase of the light traveling through the waveguide, and interference with a reference light beam (hence interferometry) is needed to measure the phase change reliably and precisely. In contrast, BLI analyzes the interference pattern of white light reflected from two surfaces: a layer of protein immobilized on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip can cause a shift in the interference pattern that may be measured in real-time as an increase in optical thickness at the biosensor tip; this results in a wavelength shift in the interference pattern.
Can GCI detect conformational changes?
Hypothetically, the Creoptix WAVEsystem can detect conformational changes, provided those conformational changes make sufficient contribution to a change in refractive index. The WAVEcontrol software also supports suitable interaction models which account for conformational changes. Despite this, conformational changes are difficult to infer purely based on either Creoptix WAVEsystem kinetic data or SPR data. This is because conformational changes are seldom a one-step process, meaning models that would perfectly fit the kinetic data would be far too complicated to be fully trusted. Additionally, conformational changes might generate unexpected responses (e.g. negative curves) due to the surface reorganization, which could prove extremely difficult to analyze and quantify consistently. Creoptix recommends performing orthogonal validation of any suspected conformational changes and ensuring that kinetic analysis is as simple as possible, for instance by analyzing kinetic differences between functional mutants.
Can I perform concentration determination experiments with the Creoptix WAVEsystem?
Yes, the Creoptix WAVEsystem can be used for quantification studies. However, the limits of detection and quantification will depend largely on the assay conditions as well as on the type and characteristics of the interaction.
How often can I use the WAVEchips?
This depends on the type of chip, the immobilization method used, and the behavior of the immobilized ligand. For example, His-tagged ligand proteins captured on a Ni-NTA surface can be regenerated by EDTA and the ligand freshly re-captured multiple times. This means that such a chip can generally be used repeatedly, provided it remains inserted in the Creoptix WAVEsystem. Regenerations are possible with both Ni-NTA and Protein A/G chips, allowing for multiple uses, however this is not easily accomplished with streptavidin or other chips.
How much ligand can you immobilize on the chips?
The immobilization capacity depends on the chip type and immobilization method. The highest capacity chip in the Creoptix WAVEsystem portfolio, the 4PCH WAVEchip, can immobilize up to 35,000 pg/mm2 of BSA, but the experimental capacity depends on the exact ligand used. The WAVEcontrol software has a built-in simulator that helps determine the ligand density required for an acceptable theoretical response.
How are the transition times of the Creoptix WAVEsystem so fast?
This is how fast transition times look like
Despite having no microvalves, the microfluidic design of the WAVEchips is fully optimized for fast transitions. Before injection of the analyte onto the sensor surface, the Creoptix WAVEsystem can prepare the sample at its full concentration near the sensor, thereby avoiding dilution effects and enabling almost immediate transitions between running buffer and the actual sample. As a result, the Creoptix WAVEsystem can resolve off-rates up to 10 s-1.
The Experimental Conditions
What is the minimum pick-up volume of the WAVEsampler (autosampler)?
The minimum pickup volume is 25 µL. However, in a typical kinetic setup, due to the dead volume of 50 µL, the minimum pickup volume is 51 µL.
What is the sample consumption of the Creoptix WAVEsystem compared to Cytiva’s Biacore & ForteBio’s Octet systems?
The Creoptix WAVEdelta has comparable sample consumption to the Cytiva’s Biacore systems (T200 and S200), while the Creoptix WAVE has a dead volume (volume needed but not injected on the surface) of 50 µl that can be lowered to 15 µl when not performing kinetic measurements. The injected volumes are determined by flow rate multiplied by injection time, as on the Biacore, and are usually dictated by the assay. For quick binders, the per-injection volumes can be <70 µl per injection, while for captures the volumes can be as low as 30-40 µl per injection. In comparison, the ForteBio’s Octet RED96e and Octet QKe report sample volumes of 180-220 µl/well.
How many experiments can I run in a day?
This depends largely on the nature of the experiment and the experimental goals. Standard workflows execute one kinetic experiment a day, beginning by setting up the assay with the necessary reagents and conditioning the chip to immobilize the ligand molecules. A typical experimental workflow involves ligand immobilization followed by a kinetic series with at least eight analyte concentrations in duplicate performed in a day. As the system allows for queueing up several kinetic series, and all the necessary analyte dilutions and reagents can be prepared in sealed 96- or 384-well microtiter plates in the autosampler, the kinetic measurements themselves can be performed hands-off overnight or even over the weekend without the need for supervision.
Are the ligand capture and immobilization techniques used for SPR/BLI also suitable for GCI?
Yes, standard immobilization techniques such as amine-coupling, Ni-NTA capture and streptavidin-biotin capture are also available for the Creoptix WAVEsystem on polycarboxylate surfaces; dextran surfaces can be supplied on request. Additionally, there is a wide range of other immobilization methods, including lipidic interactions or Protein A/G capture. An overview of the available surfaces (WAVEchips) can be found here.
How much protein do I need for a concentration determination experiment with the Creoptix WAVEsystem?
For ligands, Creoptix recommends 500 µL at 10-50 µg/mL, preferentially from a concentrated stock (50-100x). For analytes, 2 mL at 10x the KD (at minimum) can be enough for performing kinetics in duplicates.
How do I choose an appropriate immobilization/capture strategy for protein binding?
This depends on the ligand you are immobilizing / capturing. Biotinylated and His-tagged ligands can be captured on STA and NTA chips respectively. Alternatively, a suitable surface can be created for capture via whichever tag is present (e.g. coupling an anti-FLAG antibody to capture a FLAG-tagged ligand). Where there are no tags, amine-coupling or antibody capture is an option. Sometimes multiple approaches are required to achieve the best results.
Generally speaking, IgG antibodies can be directly captured on PAG chips, while lipid-particles (e.g. liposomes) can be directly captured on LIP chips. Natural membranes (e.g. VLPs) can be captured by many different methods.