The challenges of Kinetic Analysis
Conventional and established bioassays, such as enzyme-linked bioabsorbant assay (ELISA) or bead-based flow cytometry, use labeled reagents to detect and monitor biomolecular interactions. An immobilized ligand is used to capture a target analyte from the solution and then a specialized detection reagent, usually an enzyme or fluorescent dye, is used to give a measurable readout.
Since the readout relies on additional, artificial manipulation of the assay components, label-based assays can only be used to measure an analyte’s interaction at a predetermined concentration and fixed point in time. This means that kinetic analysis isn’t possible.
In addition, the labels themselves can introduce issues. They are often not inert and can directly alter the molecular structure or function of the analyte, or create an unwanted background signal by interacting, non-specifically, with other bioassay components. This can result in skewed, unreliable and poorly reproducible results.
Unwanted interactions are a particular issue when working with complex samples, such as cell culture supernatants, serum or plasma, where a wide range of, sometimes unknown, additional components may be present.
How does Label Free Characterization work?
The label-free ligand immobilization is typically achieved by covalently coupling the ligand to the biosensor using naturally occurring amine, thiol or aldehyde groups attached to the biosensor surface. Using this technique, the ligand is immobilized in its natural form.
Alternatively, the capture approach can be used for protein immobilization, taking advantage of a high-affinity interaction between the ligand and a capturing molecule that has been covalently immobilized on the surface.
Impedance or optical biosensors are then used to measure the analyte associating and dissociating with the ligand, giving valuable information about the kinetics and affinity of the binding interaction. Crucially, this is done in real-time, without artificial manipulation of the assay components.
What are the advantages of Label Free Characterization?
Because label-free interaction quantification is a dynamic and real-time technique, it opens a whole new realm of evaluation. Kinetic and affinity analysis can be explored in real-time, alongside evaluation of the binding specificity, providing more depth to the insight and answering a greater number of scientific questions, such as:
- What affinity do the molecules have?
- What is the binding kinetics between the two molecules?
- What is the biologically active concentration of an analyte in a sample?
Following its integration in January 2022, Creoptix is now a Malvern Panalytical brand. The Creoptix WAVEsystem helps researchers make discoveries that simply aren’t possible with conventional bioassay methods. High-quality data can be derived from even the most challenging sample types with superior resolution and robustness.
The power behind this system is in the patented GCI technology and non-clog WAVEchip microfluidics systems.
Conventional bioassays have relied on enzyme or fluorescent labels to detect target analytes, but these methods are fraught with non-specific binding issues that can skew results.
By removing the labels, we enable real-time monitoring without artificial manipulation.
Creoptix® WAVEsystem uses innovative and patented grating-coupled interferometry (GCI) technology to:
- Expand assays and enable dynamic, real-time kinetic and affinity analysis
- Analyze sensitive, small and weakly interacting analytes, even in complex matrices
- Automate assays for rapid throughput and high reproducibility
Download our Kinetics Guide for full details on techniques, applications, experimental design and data analysis with Creoptix® WAVEsystem:
Innovative and patented GCI technology
Grating-coupled interferometry (GCI) is a method based on waveguide interferometry and measures the changes in the refractive index within the evanescent field near the biosensor surface. Kinetic rate parameters, affinity constants and the analyte concentration can all be measured through the interaction of the analyte with the ligand and its displacement of the buffer.
GCI is unique in its ability to produce a very high signal-to-noise ratio and its capacity to deliver highly sensitive results. Unlike other surface-based, label-free technologies, such as surface plasmon resonance or biolayer interferometry, GCI measures the evanescent wave across the entire sensor surface and is not affected by environmental effects, such as temperature or vibrations, allowing for more sensitive measurement.
WAVEchip, for non-clog microfluidics
The WAVEsystem’s microfluidics cartridge, WAVEchip, supports rapid, non-clog analysis of analytes even in very complex matrices. Crude samples, pathogenic assays, harsh solvents and large particles (up to 1000 nm) can be analyzed with ease, something that can usually only be achieved with plate-based assays.
The innovative design includes all microfluidics pathways on one disposable cartridge and is simply loaded into the system for automated label free quantification. The cartridge design allows ultra-fast transition times of 150 ms for reliable determination of off-rates of 10 s-1, enabling the kinetic study of weakly binding fragments.
Making sense of the sensorgram
For more information on the WAVEsystem and its associated technology – download our WAVEsystem brochure
By observing the analyte-ligand interactions in real-time, using GCI technology, the kinetic association rate constant (ka) and kinetic dissociation rate constant (kd) can be established. Using these constants, the equilibrium dissociation constant (kD) can be easily computed. kD gives the analyte concentration when half of the ligands are occupied and this reveals the affinity of the interaction.
This information is displayed on the sensorgram and shows the binding response over time.
Typical sensorgram, showing the baseline, association, dissociation and return to baseline through regeneration.
Once the buffer is in a steady flow to create a baseline, the analyte is added and the association phase is measured (from t=0). As the analyte binds to the ligand, the refractive index of the medium next to the sensor surface increases and the signal magnifies, allowing the WAVEcontrol automated software to calculate the ka of the interaction. The steady/equilibrium state is reached once the association and dissociation rates are equal and then the running buffer is added to determine kd. Together these results give a full characterization of the interaction between the analyte and the ligand in real-time, without the need for follow-on manipulation of the assay components.
For more information on the WAVEsystem and its associated technology, download our WAVEsystem brochure.
Applications of Creoptix WAVE
With its high sensitivity, automated operation, high throughput analysis and versatility for a wide range of complex matrices and sample types, the WAVEsystem is well-suited to many applications:
Kinetic and affinity analysis
- Multi-cycle kinetic analysis
- The curvature of the association and dissociation curves on the WAVEsystem sensorgram give an abundance of information on the kinetics of the binding interaction between the analyte and the ligand. The signal level can be directly correlated to the molecular weight of the analyte, the ligand molecular weight and surface density and, therefore, the affinity of the interaction. By using a model equation and different analyte concentrations, kinetic and affinity values can be extracted and used to characterize and quantify the molecular interaction.
- Equilibrium analysis
- At the equilibrium state, the WAVEsystem can be used to gain deeper insight into weak interactions between the ligand and analyte, providing further insight into these hard-to-detect and hard-to-decipher interactions.
- waveRAPID analysis
waveRAPID is a high throughput quantification technique, characterizing large numbers of analytes, in a short period, within a wide range of properties, using repeated analyte pulses of increasing duration (RAPID). Since this robust method requires only a short analyte-ligand contact time, kinetic information can be gathered even from challenging analytes, such as non-specific binding or aggregation samples.
- Regeneration-free kinetics
For high-affinity analytes or other tight interactions with slow dissociation, regeneration-free kinetics enables the full kinetic profile to be analyzed. This method is also useful when suitable regeneration conditions can’t be found.
The WAVEsystem enables the identification and ranking of molecules that bind to particular targets from large molecular libraries. Hits are identified based on their responses which, in turn, are linked to molecular weight and the affinity of the analyte to the target ligand. The WAVEcontrol software corrects and normalizes the responses so that identified hit compounds can be ranked according to affinity.
- Membrane proteins
- More than 60% of modern therapeutics target membrane/membrane-associated proteins1, yet these structures are notoriously difficult to work with. They are often lipophilic and associate with lipophilic membranes so they require detergents to solubilize them which can interfere with the membrane protein and its ligand in a bioassay. When membrane proteins are packed in virus-like particles, liposomes, or other complexes, they can become large and require higher sensitivity measurements. The WAVEsystem tackles these complex structures with ease, allowing for effective screening and the high-sensitivity measurements needed to identify drug targets.
- Fragment-based drug screening
- As we explore new drug targets with a greater array of small molecules and fragment-based active pharmaceutical ingredients, screening requires even greater levels of sensitivity. Fragments have very low molecular weights and the size ratio to drug targets is high.
- By using GCI technology, where the light travels throughout the waveguide and creates an evanescent wave that spans the entire length of the sensor surface, more binding events can contribute to the overall signal. This gives waveguide interferometry an intrinsically higher primary sensitivity for label-free interaction analysis, enabling reliable measurements of 1:1000 size ratios.
- Since fragment-based drugs are often weak binding, the dissociation stage of label free quantification is often rapid. The WAVEchip design means that ultra-fast transition times of 150 ms can be achieved for reliable determination of off-rates of 10 s-1, enabling the kinetic study of these weakly binding fragments.
Due to its high sensitivity, the WAVEsystem can qualitatively assess the specificity of molecular interactions, ensuring no false positives through non-specific interactions. The binding specificity can be used to:
- Screen protein libraries for binding partners, followed by mass spectrometry to identify novel protein interactions
- Analyze complex matrices, such as drug-related immunogenicity in clinical trials
- Investigate specific small molecule binding interactions that might affect the pharmacokinetic profile of the drug
- Identify anti-drug antibodies (even those with weak interactions)
- Identify antibody binding sites and investigate co-factor requirements
- Use biomarkers to identify lead compounds or predict therapeutic failures
- Create diagnostic tests by capturing and detecting target antigens
Unpurified sample analysis
Biologically active and inactive species can be distinguished, even in non-purified samples, such as food, cell extracts and serum, using the non-clog features of the WAVEchip. This makes antibody analysis much easier since purification is not required following the cell culture stage and the antibody can be analyzed directly from the supernatant.
Enhancement can then fully determine antibody isotopes, without separation or washing steps. With the continuous monitoring capability of GCI, throughout the whole biosensor length, even fast-associating, low-affinity interactions can be recorded and analyzed for the deepest levels of characterization.
Are you ready for deeper insight?
With the WAVEsystem, GCI sensitivity combines with automated analytical control and innovative microfluidic technology to provide a novel, optic biosensor platform for complete, real-time, label-free binding kinetics.
Even the most challenging analytes can be quickly and easily analyzed to deliver deep insight into previously undetectable interactions.
Contact our expert team for advice on how the WAVEsystem can help you drive effective label-free quantification.