Software combo for MP-SPR Navi: MP-SPR Navi LayerSolver, 3 licenses for 1 PC each; TraceDrawer for MP-SPR Navi, 1 license for 3 PCs

In SPR, the ligand is a molecule immobilized on the sensor surface. The analyte is the molecule in the flow, and its interaction with the immobilized ligand is measured.

Immobilization is the (usually covalent) attachment of a molecule to a sensor surface. Various immobilization methods can be used, such as trapping or conjugating an amine, thiol, or aldehyde. Amine conjugation is a commonly used method, where a molecule, such as a protein, is covalently bound to the sensor surface via its amine group.

Immobilization involves at least three steps: surface activation, ligand coupling, and surface deactivation.

The volume effect is a phenomenon that occurs when the refractive index (RI) of a sample differs from the RI of the running buffer. This difference in RI is typically caused by additives (e.g., residual base solvent) in the sample. During sample injection, the change in composition causes a shift in the SPR signal, and this is called the volume effect.

The volume shift is clearly noticeable in the measurement and can interfere with the measurement of sample binding, and it occurs simultaneously with binding. In molecular interaction experiments, the volume effect is typically compensated for by reducing the response of the reference channel (no ligand) from that of the measurement channel (ligand). MP-SPR angular measurement also provides a unique internal reference for the volume effect. We call this feature PureKinetics.

Regeneration involves removing the analyte from the ligand, leaving the ligand intact and active on the surface.

Successful regeneration allows the ligand surface to be reused and another analyte to bind to the same surface. Regeneration is required for analytes that dissociate slowly. The appropriate regeneration solution depends on the interacting molecules and the type of interaction. Typically, appropriate regeneration solutions must be determined empirically.

Most often, regeneration solutions are low pH solutions, such as 10 mM glycine, or high pH solutions, such as 50 mM NaOH. Ligand activity must be confirmed after regeneration to ensure reliable results.

The selection of an appropriate carboxymethyldextran (CMD) sensor depends on the size of the analyte molecule.

If the analyte is a low molecular weight compound (e.g., a low molecular weight drug), the amount of ligand immobilized on the surface should be larger to obtain a sufficient signal. In this case, the ligand should be immobilized on the surface of the 3D CMD sensor to obtain more analyte binding sites on the surface.

If the analyte is a larger molecule (e.g., an antibody), the ligand should be immobilized on the 2D CMD surface.

A good rule of thumb is that if the analyte is less than 20 kDa, a 3D surface should be used.

Changing the buffer changes the refractive index at the surface, which causes a shift in the SPR signal.

Long measurements in an open cuvette will cause solvent evaporation and thus an increase in its concentration, which should be avoided. Close open buffer flasks, and for long experiments, cover 96-well plates and sample vials with appropriate lids.

It is very important to always clean the glass side before inserting the sensor into the instrument. When the flow cell is closed, the prism is tightly connected to the glass side of the sensor slide via an elastomer with a matching index. If the glass side is not clean, particles will adhere to the prism elastomer and interfere with measurements.

The glass side is cleaned gently by wiping it with KimWipes (lint-free paper) moistened with ethanol or isopropanol.

The gold sensor can often be completely cleaned by oxidation (using e.g. ammonia and H2O2) and the sensor is usually recyclable multiple times.

Hydrogel-coated sensors cannot be cleaned without removing the dextran coating.

The correct cleaning procedure depends on the sensor coating.

Typical SPR sample volumes for all biochemical interaction experiments are 100 to 300 µl (up to 500 µl in the semi-automatic SPR Navi 200 and MP-SPR Navi 200 OTSO models).

Please note that the amount of sample needed per injection is actually concentration x volume!

A wide range of different coatings are available, suitable for both covalent and non-covalent protein conjugation.

We offer sensor functionalization for all of the above methods.

MP-SPR works well with many lipid forms. It offers the advantage of being able to check the conformation of the formed layer before injecting the protein. Based on its thickness and refractive index, it is possible to verify that the film is properly formed and is indeed a bilayer/liposome layer. For supported lipid bilayers, we recommend our SiO₂-coated sensors. For liposomes, the CMD sensor is recommended.

MP-SPR measurements have also been performed on lipid-binding sensors. The sensor has a three-dimensional hydrogel surface and lipophilic anchor groups that capture liposomes through nonspecific interactions. Other methods include the use of biotinylated liposomes, His-tagged proteins embedded in liposomes, DNA fragments embedded in liposomes, or silica surfaces (which self-adsorb liposomes).

The standard flow cell material is PDMS and is compatible with, in addition to aqueous buffers, e.g., glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide).

Highly chemically resistant flow cell materials include PEEK and Kalrez, and a much wider range of solvents can be used with them.

The MP-SPR Navi 200 OTSO also uses PDMS as the standard peristaltic tubing material. An external syringe pump can be used if other organic solvents are required.

A complete list of compatible solvents and vessels can be found in the appendix of the MP-SPR Navi user manual.

Experiments can be performed with soluble and membrane proteins as well as proteins embedded in lipid vesicles.

The standard configuration will work with materials with a solid-state RI from n=1 to n=1.4, and even up to 1.45 when multi-wavelength systems are used.

If an even higher RI is required (for certain organic solvents), BioNavis offers a separate high RI configuration. The RI of the measuring liquid can then be up to n=1.51.

The most common problems with liposome deposition are due to surface and instrument cleanliness or changes in liposome size.

To achieve consistent lipid bilayer formation on the SiO₂ SPR sensor, cleaning before each lipid deposition is essential. An effective cleaning protocol involves using a CHAPS-Helmanex-ethanol-water mixture before each liposome deposition. The sensor should be used immediately after cleaning prior to deposition.

Hydrophobic layers can cause air to form or be trapped in the flow cells. This air bubble will prevent good liquid-to-surface contact, negatively impacting SPR signal quality.

If an air bubble becomes trapped on the surface, consult your device's user manual for instructions on how to remove it. To prevent bubble formation, carefully degas buffers and be careful not to introduce air bubbles during sample injection.

EDC/NHS coupling typically binds a large number of proteins, but the binding conversion efficiency is higher than that of 100%. Stabilization (injection) after immobilization is necessary to remove unbound, adsorbed protein from the layer, which may negatively impact the experiment in the subsequent analysis phase.

Sometimes, after regeneration injections, a stabilization period (several minutes) is required when using the CMD-3D sensor. Regeneration solutions typically exhibit significant chemical differences from the measurement buffer and can cause hydrogel swelling/shrinkage or salt adsorption/desorption, leading to a small but systematic drift. In such cases, adding a few extra minutes to the baseline time between measurements is a good idea and will save time later in the analysis.

Deionized or purified water is recommended for buffer preparation. Filtered liquids, buffers, and samples help prevent the suspension from accidentally adhering to surfaces and affecting the SPR signal. Degassing solutions will also contribute to more stable results.

BioNavis LayerSolver software is software specifically designed to calculate the thickness and refractive index of a layer.

Typical procedure based on the measurement of two wavelengths:
1) Measurement of the cleaned sample surface at two wavelengths simultaneously
2) Measurement of the deposited layer at two wavelengths
3) Analyzing both wavelengths simultaneously using LayerSolver
4) The dn/dlambda (RI wavelength increment) is required for the given material or similar type of materials.

Initial parameters for BioNavis gold sensors and 670 nm wavelength:

The minimum SPR peak angle can be roughly converted to 10 RU, or 1 ng/cm² at 785 nm. The constant intensity angle depends on the sensor surface area. These values can be converted to surface protein concentration (moles/cm² = surface amount) if the molecular weight of the proteins is known (using the formula: Amount = Mass/Molar Mass, n = m/M). Surface coverage scaling can be enabled in the "Scaling Options" in both Viewer and Control software.

It's important to remember that this is an approximation (though widely accepted) and only works for proteins. This is because the optical properties of proteins are similar in most cases. For more exotic samples, knowledge of the RI (mass) vs. concentration (dn/dc) relationship (from the literature or measurements) is necessary to obtain an accurate conversion.

Below is the full conversion of SPR peak angle change to surface concentration in moles. Note that because protein molecular weight is taken into account, this formula is not actually the same for different proteins, but varies slightly for each one.

Amount (moles/cm²) = (SPR angle (degrees)*1000*10^-9 (g/cm²/degrees))/ protein molecular weight (g/mol)

You can leave the device on all the time if you plan to use it every day.

However, if you don't need to use the device every day, it's better to turn it off to save energy. If the power line is not stable enough or you experience occasional power outages, please add a UPS (uninterruptible power supply) to the device.

The MP-SPR Navi should be placed on a stable table. The device is very sensitive to mechanical shock, so avoid exposing it to vibrations that could disrupt measurements. Select a location where the device is not exposed to external heating or cooling sources, such as direct sunlight, near vents, or air conditioning units. External heating or cooling can cause temperature fluctuations and disrupt measurements.

Suitable storage conditions for BioNavis systems:

Temperature +15°C – +30°C, stable temperature.
Relative humidity from 25% to 60%.
Low dust, ISO-9 class room according to ISO-14644-1 standard ("room air" conditions according to US FED STD 209E standard)
Stable and grounded power source (USP recommended)
Keep away from direct sunlight
Keep away from sources of ignition
Keep away from direct drafts such as fans or air conditioners

The maximum injection time depends on the flow rate and loop volume.
If the instrument contains 250 µl loops and the flow rate is 30 µl/min, the maximum injection time is 7 minutes.
It is recommended to only inject 80% loop volumes when concentration is critical for measurement, as the sample tail will be diluted (depending on the instrument model). In automated models, the injected sample can be protected from dilution by air segments. Watch an animation and learn how flow injection works here.

Also remember that you do not have to inject the entire volume of the loop, you can perform shorter injections.

Once the sample is empty, the buffer will flow into the measurement channel.

The standard loop size is 250 µl, but the loop can easily be replaced with a smaller or larger one.

The automated instrument models (210A VASA, 220A NAALI, and 420A ILVES) allow for partial loop injection, which allows the loop to be filled with less sample than its total volume. This is a very useful feature when only a small amount of sample is available and does not require a loop change.

Yes, BioNavis offers the ability to measure SPR and fluorescence signals simultaneously.

There are three options available:

• fluorescence coupled to surface plasmons (option 1)
• beam splitting fluorescence (option 2)
• fluorescence of fiber bundles (option 3).

The BioNavis device is flexible and user-friendly. Therefore, we do not mark channels in the injection ports.
In MP-SPR Navi 200 OTSO the standard connection is as follows: left the injection port is connected to upper channel, and the right injection port to the lower flow channel.

However, there is an easy way to check this:
Once all the lines are dry, insert the gold sensor into the device and begin angular measurement. Fill one (!) of the channels with approximately 10% ethanol or water, and leave the other injection loop empty (air). Start the pump (no buffer, just air) with the injection valve set to "injection." Check which channel changes the signal. Remove the sensor and check which channel is wet.

When the flow chamber is closed, the prism is tightly connected to the glass side of the sensor slider via an elastomer with a matching index. If the flow chamber is closed for a long time, the elastomer may become damaged. A damaged elastomer would interfere with measurements.

You can check if the elastomer is in good condition by observing it with the naked eye for scratches, particles, dried salts, or by testing it.

Place the glass sensor (without coating) in the instrument and perform a full angular scan. The curve should increase to approximately 1° and remain flat. If this does not happen, the prism elastomer can be wiped with isopropanol (see the appropriate instrument's instruction manual, "Troubleshooting" section) and the instrument calibrated. If calibration does not resolve the issue, the prism will need to be replaced.

Note that spatial resolution is typically quoted for instruments that use a CCD camera as the detection component, such as SPR imaging. These instruments are typically limited by the camera resolution and do not offer as good sensitivity as focused beam or MP-SPR instruments.

In our case, the detection principle is based on a true goniometric configuration. The actual angular resolution of the goniometer is 0.001 degrees (the smallest goniometer step), but because we use advanced peak detection algorithms to find the minimum, the SPR minimum position is actually determined more precisely than the goniometer step.

The diameter of the laser "spot" on the sensor plate surface is approximately 0.5 mm. The MP-SPR measurement results are averaged over this area (i.e., the thickness is an average over an area of 0.2 mm²).

If by spatial resolution we mean the detection limit of the instrument, then the resolution depends significantly on the measurement system and the analyte. The amount of signal produced by the analyte can be increased by the sensor structure (three-dimensional hydrogels instead of two-dimensional monolayers), and for three-dimensional hydrogel sensors, the estimated accuracy of direct detection of low-molecular-weight analytes is in the nano-micromolar range (or approximately 300 fg/mm² – femtograms per square millimeter), BUT it depends on the interactions themselves.

The key difference lies in the optical system. Traditional SPR uses a focused beam system, which provides an angular range of several degrees and measures only a single parameter—the SPR peak minimum, which is then plotted on a sensor image. The typical RI (refractive index) range of traditional SPR is 1.3–1.4 (the RI of the liquid).

MP-SPR utilizes a Kretschmann configuration and goniometric optics, enabling a scan range of 38 degrees and an RI of 1.0 to 1.4. In addition to measuring the SPR peak, the full SPR curve is monitored and other parameters are recorded. Parameter cross-correlation enables a unique PureKinetics feature. Wavelength measurements using multiple lasers enable characterization of layer thickness and refractive index, providing insight into, for example, molecular conformation changes or layer swelling.

The MP-SPR's special optical configuration enables a much broader range of applications in life sciences, biosensor development, and materials characterization. An exceptionally wide range of sensor surfaces is also available, including Ag, Cu, Al2O3, SiO2, PET, PS, cellulose, and CaP (calcium phosphate). MP-SPR can measure thin films (e.g., a single 3.7 Å graphene monolayer) as well as thicker films (several micrometers thick, e.g., polyelectrolytes or living cells), and measurements can be performed not only in the liquid phase but also in the gas phase.

Angular scanning is the recommended measurement mode for most experiments.

In this mode, the light source scans continuously over a selected angle range, and the SPR peak (angle vs. intensity plot) can be monitored. Several SPR peak parameters can be tracked over time (e.g., PureKinetics, minimum peak position, minimum peak intensity). Thickness and refractive index can only be calculated from angle scan data!

In angle mode, the light angle is constant (the light source does not move), and the reflected light intensity is monitored as a function of time. Angle measurement allows for increased sampling rates—up to 250 points per second (4 ms sampling time), but collects less information about surface changes. In angle mode, the typical sampling rate is 2 seconds.

In angle mode, more information can therefore be collected from the measurement compared to angular measurement, so we strongly recommend using the angle scan mode.

The temperature range is +20/-7°C from ambient temperature. This is approximately 15-45°C under typical laboratory conditions.

Temperature is controlled using a Peltier element. The well plate area of the MP-SPR Navi 220A NAALI and 420A ILVES models can be cooled to +4°C.

The standard instrument is equipped with a 670 nm laser in each flow channel.

Standard instruments with an additional laser kit (option -L) are equipped with 670 nm and 785 nm lasers in each channel.
Two wavelengths per flow channel are necessary to determine the thickness and refractive index.
Higher wavelengths have deeper penetration, greater dynamic range, but lower sensitivity.

We can also provide other wavelengths, subject to availability. To date, we have installed custom wavelengths: 635, 650, 670, 785, 850, and 980 nm.

We can also provide a custom input, allowing you to use your own light source (including fluorescent). The light source must be equipped with an external trigger.