Highly sensitive analysis at low immobilization levels, without mass transport limitation

In this technical note, we show the remarkable sensitivity of the Creoptix WAVE. Thanks to the expanded sensing field over which the Grating-Coupled Interferometry (GCI) technology measures, high resolution kinetic determination at even very low responses is possible, potentially reducing material costs significantly.

Summary

During label-free analysis of molecular interactions, mass transport limitation represents a significant source of deviation from first-order kinetics. Arising from analyte concentration gradients between the bulk solution and the surface, mass transport limitation is especially pronounced at the high-density ligand immobilization levels often required to generate a measurable binding signal.

Using a proprietary Grating-Coupled Interferometry (GCI) technology, the Creoptix WAVE delivers superior sensitivity over traditional Surface Plasmon Resonance (SPR) methodologies, particularly with low ligand immobilization levels of poorly active proteins. This translates to reliable kinetics at even very responses (Rmax <1).

By characterizing the interaction between acetazolamide and Carbonic Anhydrase II, using different immobilization densities of the enzyme, we show that the Creoptix WAVE affords greatly reduced sample consumption while avoiding mass transport limitation.

CAII capture level
(pg/mm2)
kon
(M-1.s-1)
koff
(s-1)
kmRmax
(pg/mm2)
KD
(nM)
Kinetic data MTL model81001.29x1060.04186.12x10618.68832.5
Kinetic data 1:1 model12400.77x1060.0324-0.46942.3

Table 1: Kinetic data for acetazolamide binding onto different Carbonic Anhydrase II (CAII) densities

Legend: (A) Sensorgrams showing the interaction between acetazolamide (analyte, MW 222.25 Da) and carbonic anhydrase II (CAII, ligand, MW 29 kDa) at low and high immobilization densities. CAII was captured on a 4PCH WAVEchip at different immobilization densities, as indicated. Dose-response curves for acetazolamide were recorded at each immobilization level. Mass transport limitation (MTL) model was used to fit the data from the high immobilization level (left) as the interaction deviated from 1:1 model (right) using Creoptix WAVEcontrol software. (B) The data shows that the kinetic fits at the low immobilization level are not mass-transport limited, but rather follow first-order kinetics.

[Figure 1 TN210104-Creoptix-sensitive-analysis-low-immobilization-levels.jpg] Figure 1 TN210104-Creoptix-sensitive-analysis-low-immobilization-levels.jpg

Figure 1: Acetazolamide binding onto different Carbonic Anhydrase II (CAII) densities

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