Method description

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.

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Required Materials

• HexagonFab Bolt Reader (supplied)
• HexagonFab Bolt Sensor (supplied), variant A or B
• HexagonFab Bolt Software, installed on computer/laptop
• Streptavidin, purified, lyophilized powder (Sigma-Aldrich, 85878)
• Anti-Streptavidin antibody, purified (Abcam, ab10020)
• N-Hydroxysuccinimide (NHS, Sigma-Aldrich, 130672)
• 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich, commercially available)
• 1 x PBS, Dulbecco’s phosphate buffered saline (PBS, Sigma-Aldrich, D8537)
• Ultra-pure grade water (or equivalent filtered water)

Assay preparation

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:

• Equilibrate all reagents used to room temperature before solution preparation.
• Incubate 25 μL 100 μg/mL ligand solution over the exposed Bolt sensor for 1 hour at 35C (or 2-3 hours at room temperature). Once complete, wash the sensor area several times with 1 x PBS, then either dry or hydrate with 0.01 x PBS for measurement.
• For sensor variant B, activate the sensor surface with a 0.2 M EDC/0.05 M NHS solution in ultra-pure water first before ligand incubation.

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.

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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:

• Add 100 μL 0.01 x PBS: 300 seconds (s)
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 1 μg/mL Ligand in 0.01 x PBS: 600 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Set Baseline: 30 s
• Remove 100 μL, add 100 μL 13.3 nM Antibody in 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 26.6 nM Antibody in 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 39.9 nM Antibody in 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 53.2 nM Antibody in 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 66.5 nM Antibody in 0.01 x PBS: 300 s
• Remove 100 μL, add 100 μL 0.01 x PBS: 300 s

Protocols can be saved and reused for repeat experiments, or otherwise modified for experiments requiring new parameters.

• Once the protocol is entered, the measurement window shows the electrical measurement across the sensor area (in response units) against time. The experiment protocol is displayed to the left-hand side of the screen.
• Once ready to begin, the measurement reading begins with the first step in the experiment protocol, which will run for the defined time period in the protocol.
• When the step time reaches 0, the user will be prompted to move on to the next step. Once the end time of the final step is reached, the prompt will read that the experiment has concluded. All steps carried out with their step times are recorded along with the data.
• The Bolt sensor area well can hold approximately 100 μL of solution, which can be introduced or removed using a standard 100 μL capacity mechanical pipette. For general usage a volume of 10 μL - 100 μL is recommended.
• As the experiment proceeds, the experimental data is saved at frequent intervals, using the common filename from the protocol file and in the same filename location. At each protocol step also, the step name and time duration are also saved in a notes file. This makes post-experiment data visualisation simple for the user.
• During the experiment, extra notes can be added manually by the user, useful in case the experiment deviates from the experimental protocol for example.
• Once the experiment is complete, the data will be automatically saved. After measurement, the program can be navigated back to the protocol window or back to the home screen, ready for another experiment.

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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.

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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).

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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.

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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.

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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.