Examining Protein Interactions with Nicoya’s Alto SPR System

Nicoya produce benchtop SPR systems; the OpenSPR, which uses Localised Surface Plasmon Resonance (LSPR) Technology and the Alto, which also uses LSPR that is integrated with digital microfluidics (DMF) and nanotechnology. You can find out more about how SPR works and its similarities to LSPR here.

The Alto

The Alto is the world’s first digital, high-throughput, benchtop SPR system which is achieved using digital microfluidics. DMF is a liquid handling technology that can accurately control nanolitre droplets. This is done using applied voltages across the system giving the instrument wide flexibility over assay design. On top of having this, in the Alto all fluidics are handled on one disposable microwell plate, which is adapted to precisely handle sample volumes of 2 microlitres. 

Image 1 shows the digital microfludics technology used within the disposable cartridge of the Alto system.

This gives the Alto a large series of advantages which include:

  • Sample friendly due to its compatibility with crude samples.
  • User-friendly automation without fluid maintenance.
  • 24+ hours unattended runtimes with simple sample-in/answer-out data.
  • Improved data quality and assay flexibility.
  • Reduced human error and time savings with automated serial dilutions.

Advantageously, the Alto can provide a variety of scientific information including Kinetics/Affinity Characterisation + Screening, Quantitation and Epitope Mapping/Binning. The data that can be obtained using this SPR system has applications in many biomolecular interactions. At Merrow Scientific we look at protein interactions and offer contact testing for these kind of interactions, but the Alto has capabilities to look at the interactions of Antibodies, Nucleic Acids, Carbohydrates, Crude Samples, Viruses and Small Molecules.

Protein-Antibody Interactions

An example of an application of protein interactions was the comparison of single-sequence and multi-sequence kinetic analysis of a protein-antibody interaction which was undertaken by Nicoya on their Alto, where they obtained binding kinetics and affinity data. This comparison was undertaken by analysing Protein A with Human Immunoglobulin G (HIgG) through both kinetic methods. Kd for this interaction was determined to be 1.93 nM and 3.39 nM with single-sequence and multisequence kinetics, respectively. This shows highly comparable results can be obtained for a given interaction with these different assay methods.

Kinetic titration, also known as single-sequence kinetics is a SPR method where the analyte containing solution is passed over the immobilised ligand in sequence of increasing concentration. This process also only has one dissociation and regeneration phase, which takes place after all the analyte concentrations have bound to the ligand on the sensor.

Image 1: Single-sequence binding of HIgG at 300 nM, 100 nM, 33 nM, 11 nM, and 3.7 nM to the immobilized Protein A. The solid black line represents the one-to-one kinetic model fit with global Rmax .

Multi-sequence kinetics is a type of analysis that involves alternating the cycles of analyte injections and surface regeneration. This means each concentration is treated as a separate sequence, so this method produces multiple SPR curves compared to the single curve produced by SSK.

Image 2: Multi-sequence binding of HIgG at 300 nM, 100 nM, 33 nM, 11 nM, and 3.7nM to the immobilized Protein A. The solid black lines represent the one-to-one kinetic model fits with local Rmax.
Image 3: Full trace of the MSK assay showing the return to baseline after each regeneration. Full regeneration is not achieved for higher analyte concentrations. A local Rmax was used in the kinetic fit to account for this.

Both these kinetic methods have their own set of advantages and disadvantages, but Alto can run both, allowing for comparison and to see which method best suits the interaction you are looking to study. This shows the instruments ability to facilitate both assay methods easily and accurately. More information on the application note can be found here and other SPR Protein interactions experiments undertaken using Nicoya Instruments can be found here. If you have any questions or are interested in SPR contract testing please contact us.