The HexagonFab Bolt sensor is designed to measure the binding interaction between an antigen and antibody in a buffer solution when connected to the HexagonFab Bolt reader. In this case, the sensor is first loaded using commercially available Streptavidin (the ligand) dissolved in standard PBS solution. Then, the sensor is connected to the reader system and a background signal for the system is established using a 0.01 x PBS solution. Once equilibrium is reached, multiple solutions of Anti-Streptavidin (13.3 nM – 66.5 nM) in 0.01 x PBS (the antibody) are tested, with washing steps between each concentration using 0.01 x PBS. The resulting measurement, showing association/dissociation curves for each concentration, is then analysed using the included software to give association/dissociation constants for each concentration.
The sensor chips are supplied functionalised with a chemical layer which is used to anchor the target protein to the graphene surface. There are two variants, A and B. In variant A the surface is terminated with aldehyde functional group, whereas variant B is terminated with carboxylic acid groups.
Depending on the sensor variant used, the sensor is either immediately reactive to amine residues on the ligand protein (variant A) or must be activated using standard EDC/NHS activation mixtures (variant B). For loading:
It is also feasible that the ligand loading can be carried out once the bare sensor has been connected to the reader and measurement has started. In this case, during the measurement stage the incubation is performed as described at room temperature until the on-screen response curve to reach saturation, which should take no more than 30 minutes. After this, the sensor area can be washed before proceeding with the binding measurement steps.
Experiment setup and measurement
Once the chip is functionalised, it is ready to be connected to the Bolt reader. Ensure that the reader is plugged in with the power on and is connected to a computer or laptop via USB with the HexagonFab Measurement/Analysis Software installed. For this experiment, the sensor was loaded with the streptavidin ligand as part of the experimental protocol.
The software includes an intuitive protocol writer, where the experiment can be planned out by defining each experimental step (usually a change in solution), along with the corresponding time interval. For this example experiment, the protocol was set as:
Protocols can be saved and reused for repeat experiments, or otherwise modified for experiments requiring new parameters.
Analysis of the Streptavidin/Anti-Streptavidin association and dissociation curves
The experiment as outlined has three experimental steps – 1) loading of the ligand, 2) washing and equilibration for baseline, and 3) association dissociation measurement for the 5 concentrations of antibody studied. It is recommended that a negative control is run, either using 0.01 x PBS or 0.1% Bovine Serum Albumin (BSA), to verify the results of the experiment. An example of the resultant data from the experiment is shown below, with experimental step times highlighted.
For analysis of the resulting curves, the HexagonFab Bolt software includes an analysis package, accessed from the home screen by clicking the Analysis Tool button. The data file generated from the experiment can be opened and displayed. To perform an analysis on the curve, set the baseline, association and dissociation sections of the curve you wish to measure defined by time interval (for example the baseline for the start of the 13.3 nM curve starts at T = 2300 seconds).
Three Baseline Fit Methods are included in the dropdown box: Linear, Exponential Decay and Fractional. This is for the purposes of baseline correction for the association and dissociation curves, and which function should be used depends of the baseline shape (for example, how straight or curved the baseline is before association). In this specific experiment, Exponential Decay was chosen.
Once all parameters are set, the analysis tool cuts the graph at the defined ranges, then fits the baseline and applies a correction to the association and the dissociation. Then, these curves are fit using a built-in biophysical model, from which the program calculates the association rate constant (ks) and the dissociation rate constant (kd) for the ligand/antibody interaction. Once calculated, the results are displayed in a new pop-up window and automatically saved in PDF format. The data for the smoothed curve sections are also saved as CSV files for further data manipulation if required.
The analysis steps can then be repeated to study the other curves in the data set and get a full set of association/dissociation curves with corresponding ka/kd values.