Identification and analysis of particle-particle, particle-biomolecule, functionalisation and aggregation interactions.
Particle interaction monitoring
Background
The Izon SIOS technology can be used to detect, analyse and monitor interactions between nanoparticles or particles and biomolecules. Binding reactions can result in the generation of products that exhibit differing electrophoretic properties to that of the original constituent particles. Such interactions may result in a measurable change in key SIOS measurement parameters; blockade event frequency, duration and magnitude.
This can be used to:
- Confirm the occurrence of a binding interaction or particle functionalisation
- Compare properties of samples prior to and post interaction
- Detect the presence of molecules that may otherwise be below the detection limit of the instrument
- Monitor the binding interaction event in real-time particle-by-particle by combining the reactants in the top fluid cell
Changes in blockade event magnitude reflect a change in the size of the particle and its associated ion cloud. Changes in blockade event duration or frequency reflect an alteration in the particle's mobility. Changes in mobility may indicate variations in the charge of the complex when compared to its precursors, affected through alteration of the surface properties of the particle. These manifest themselves as variations in hydrodynamic friction and electrophoretic retardation through the influence of the applied voltage on the ion cloud surrounding the particle.
Suitable interactions that might be studied using the SIOS technology platform include those between antibodies and an antigen, the binding of ligands to receptor complexes, particle-particle aggregation and nonspecific adsorption of solutes onto particle surfaces.
To observe particle interactions, the components may be preincubated and, after a set time, analysed using the SIOS technology. Alternatively, the reactants can be combined in the upper fluid cell of the instrument and the electrophoretic properties of the interaction analysed in real-time.
The SIOS technology platform is capable of identification and analysis of binding interactions between particles and biomolecules
Confirmation of particle interaction or functionalisation
An interaction event between two particles (including particle functionalisation) may exhibit as a change in the measured blockade event frequency, duration or magnitude. Measurement of this change can be used as confirmation of an interaction.
This concept is demonstrated in the example on the left by considering the interaction of avidin molecules (~ 3 x 4 x 6.5 nm) with carboxylated polystyrene particles (mean diameter 184 nm). The carboxylated polystyrene provides a highly negatively charged surface for the positive avidin to bind to. The figures demonstrate that both blockade event frequency and duration as measured by the system are affected by the binding interaction. In the first case a relatively high concentration of avidin is added to the sample and the surface of the polystyrene particles become completely coated with avidin molecules resulting in reduction of the blockade event frequency to zero. In the second case a lower concentration of avidin was added to the sample, partially coating the surface of the polystyrene particles, modifying the net surface charge, resulting in an increase in the measured blockade event duration.
Confirmation of interaction between avidin molecules and carboxylated polystyrene particles. Demonstrated through changes in both the frequency of recorded blockade events and the measured mean duration of the blockade events for two different concentrations of avidin.
Sample comparison
Analysis of the key measurement parameters, blockade event frequency, duration or magnitude can provide information about differences in properties of precursor particle samples and the post-interaction complex. One area of application is monitoring the blockade event magnitude to detect and analyse the presence of aggregated particles in a sample following interaction. In the example to the left note the population of large magnitude blockade events in a sample of carboxylated polystyrene to which 50ng/µL of avidin molecules have been added. The avidin molecules have bound together some of the polystyrene particles in the sample, resulting in large aggregates. The relative size of the aggregate particles to the single particles in the sample as well as their concentration as a volume fraction of the sample may be gleaned from the data by studying the magnitude histogram results and translating this data into relative size values using a volume approximation.
The presence of a large amount of avidin causes the polystyrene particles in the sample to aggregate. The aggregated particles have a larger measured blockade magnitude and duration that the single particles, clearly demonstrated in the second figure and in the calculated mean values.
Real-time monitoring of interactions in situ
Particle-particle interactions can be carried out in situ in the upper fluid cell and the interaction monitored in real-time through changes in SIOS measurement parameters. In the example to the left 10ng of avidin was added to 184nm carboxylated polystyrene particles in situ in the upper fluid cell. A clear increase in the measured mean blockade duration can be seen following introduction of the avidin.
A binding interaction event (in this case avidin and polystyrene) is monitored in real-time after the reactants are combined in the upper fluid cell of the instrument.
Small molecule detection and diagnostics
Molecules that otherwise may be too small to be detected by the SIOS technology presently may be detected indirectly by monitoring key blockade event parameters. The examples in this page demonstrate the use of caboxylated polystyrene to detect the presence of avidin through interaction monitoring as changes in measured blockade frequency (Interaction Confirmation), blockade duration (Interaction Confirmation and Real-time Monitoring) and blockade magnitude (Sample Comparison). This principle may be used as a diagnostic tool using specific functionalisation of nanoparticles to detect molecules of interest in a sample through changes in blockade event parameters caused by specific binding of the target molecules to the nanoparticle surface.
Variation in blockade event parameters due to specific binding of target molecules to functionalised nanoparticles provides the potential for a targeted diagnostic solution.