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Instructions
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Azide-modified Sensor Chips (PDF download)

Product description

Product code Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD +
Add: AZHC30M, AZHC200M, AZHC1000M, AZHC1500M, AZD50L, AZD50M, AZD200L, AZD200M, AZD500L, AZD500M, AZD700L, AZD700M
Example: SCBS AZHC200M
Intended purpose Azide-modified sensor chips are designed for covalent ligand immobilization of DBCO-modified biomolecules via a highly selective click reaction. For successful immobilization, the ligand must contain at least one reactive primary amino group, a DBCO-functionality or a comparable click compatible functional group. Recommended applications include the investigation of biomolecular interactions involving proteins, nucleic acids and small molecules.
Storage Store at -20 °C, desiccated over molecular sieve 4A
or at 2–8 °C in physiological buffer.
Related products
  • DBCO-labelling kit, product code K DCL-3C
  • Coupling buffers (acetate or maleate) pHs 4.0–6.0, product codes B A40-50ML, B A45-50ML, B A50-50ML, B A55-50ML, and B M60-50ML
  • Borate elution buffer, product code B BELU-50ML

Introduction

Click Chemistry has gained increasing popularity among chemists due to its fast reaction kinetics and excellent selectivity. To make this unique bioconjugation technology available for SPR biosensing, XanTec offers azido (N3)-derivatized polycarboxylate sensor coatings, which immobilize cyclooctyne (DBCO)-labelled ligands in a fast and selective manner. Using a procedure very similar to ligand biotinylation, DBCO can be conjugated with a variety of ligand molecules.

The surface-bound azido groups generally exhibit high bioinertness due to their zwitterionic character, contributing to the overall bioinertness of the sensor chip.

Unlike EDC/NHS chemistry, DBCO/azide coupling is not time-critical; both reaction partners exhibit exceptional in-situ stability for up to weeks within a pH range of 4–10. Additionally, the high selectivity of both reaction partners minimizes the risk of unwanted side reactions, eliminating the need for quenching steps. As a result, immobilization via DBCO/azide coupling is highly reliable, convenient, and flexible. Due to the fast reaction kinetics, immobilization under physiological conditions is feasible, though immobilization densities may be somewhat lower. Therefore, sufficient electrostatic preconcentration remains a prerequisite for high immobilization densities of proteins, making preconcentration scouting of the ligand (see protocol below) advisable before the actual immobilization. Small molecules, however, can freely diffuse into the sensor matrix and do not require electrostatic preconcentration for efficient immobilization.

An optional labelling kit provides all necessary reagents to perform three DBCO–protein modifications for subsequent immobilization on azide-modified sensor chips.

Azide-modified Sensor Chips
Fig. 1: Controlled ligand immobilization in five successive immobilization cycles of 2.5 µg/mL DBCO-modified protein A/G on an AZHC30M chip and 2.5 µg/mL unconjugated protein A/G on an EDC/sNHS-activated HC30M chip (reactions in 5 mM acetate buffer, pH 4.5).

Additional materials required for Click coupling

Coupling buffer (dependent on the pI of the ligand):
Acetate buffer pH 4.0 (product code B A40-50ML): 5 mM sodium acetate, pH 4.0, 50 mL
or Acetate buffer pH 4.5 (product code B A45-50ML): 5 mM sodium acetate, pH 4.5, 50 mL
or Acetate buffer pH 5.0 (product code B A50-50ML): 5 mM sodium acetate, pH 5.0, 50 mL
or Acetate buffer pH 5.5 (product code B A55-50ML): 5 mM sodium acetate, pH 5.5, 50 mL
or Maleate buffer pH 6.0 (product code B M60-50ML): 2.5 mM sodium maleate, pH 6.0, 50 mL

Ligand bearing DBCO-group (to be provided by the user)

Borate elution buffer (product code: B BELU-50ML): 0.1 M sodium borate, 1 M NaCl pH 9.0, 50 mL

Optional: DBCO-labelling kit (product code K DCL-3C): Use for DBCO-conjugation of biomolecules which contain at least one reactive primary or secondary amino group.

Preparations for Click Coupling

Clean the SPR fluidics

Ensure that the flow system of your SPR equipment is free from any protein contamination, as even small amounts of desorbed protein can accumulate on the charged sensor surface. If necessary, clean the system using either 1 % Tween 20 or, for a more stringent cleaning, 0.5 % SDS for 5 minutes, followed by 50 mM glycine·HCl (pH 9.5) for 10 minutes (both included in the Desorb Kit, product code K D-500ML). The glycine is required to remove residual traces of SDS.

Sensor chip

Allow the sealed sensor chip pouch to equilibrate to room temperature to prevent condensation on the chip surface.

After opening the pouch, install the sensor chip according to the instrument manufacturer’s instructions.

Note: XanTec SPR sensor chips, like all nanocoatings, are prone to degradation when exposed to the atmosphere due to reactive oxygen species in the air. To prevent this, unmounted sensor chips should be stored in a closed container under an inert gas atmosphere or in a physiological buffer for short-term storage.

Optional: Scout for electrostatic preconcentration conditions

If the pI of your protein is known, a coupling buffer with a pH 0.5–1.0 units below the pI of the ligand is recommended for efficient immobilization. Protein concentrations of 1–100 µg/mL are usually sufficient for efficient covalent click coupling of DBCO-modified proteins.

If the pI of your protein is unknown, you may want to perform an electrostatic preconcentration scouting. In this case, dilute your non-conjugated protein stock solution into different coupling buffers with final protein concentrations of 5–25 µg/mL. Start at pH 6.0 and decrease the pH in steps of 0.5 until pH 4.0.

Please note that preconcentrated DBCO-modified protein instantly reacts with the surface bound azido groups and thus cannot be removed afterwards. It is therefore advisable to perform the electrostatic preconcentration scouting with the non-conjugated protein.

Procedure for electrostatic preconcentration scouting Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with physiological running buffer and mount a compatible XanTec sensor chip.
2 Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. 25 3 × 60
3 Inject your protein (5–25 µg/mL) in Coupling buffer.
Start at pH 6.0 (maleate coupling buffer). After protein injection, wait for 60 s, then inject the next protein solution at a pH 0.5 units lower than the previous solution.
Repeat until you reach pH 4.0. Select the highest pH value that
allows a sufficiently high pre-concentration effect.		 10 600
4 Inject Borate elution buffer. Wait until the baseline has stabilized. 25 60

Optional: Ligand DBCO-conjugation

Ligand DBCO-conjugation is described in the DBCO-labelling kit product information. In general, the procedure is similar to standard biotinylation via amine-reactive NHS ester. Sub stoichiometric degrees of labelling are crucial to generate optional immobilization outcomes and ligand activities.

Protocol for Click Coupling

Procedure Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with water as running buffer and mount a compatible XanTec sensor chip.
2 Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. 25 3 × 60
3 Inject your DBCO-modified ligand solution in a suitable coupling buffer. A protein concentration of 1–100 µg/mL is recommended. Wait until the baseline has stabilized. 10 600
4 Inject Borate elution buffer to remove loosely physisorbed ligand. 25 60
5 If the immobilization level is sufficient, switch to a physiological running buffer and wait for the SPR signal to stabilize. Otherwise, repeat steps 3 and 4 until the desired ligand immobilization level is achieved.
6 Start interaction analysis. We recommend beginning with 3–5 consecutive regeneration cycles to improve data quality and stabilize the chip surface.

Notes

It is recommended starting with low ligand concentrations (1–2.5 µg/mL) and re-immobilize with higher concentrations as required. This prevents excessive immobilization on the sensor coating. Additional immobilization steps can also be conducted during the interaction experiment.

Physiological running buffer can be used instead of water during immobilization. Make sure that the buffer is free from sodium azide.

Immobilization in physiological coupling buffer, although less efficient, can be a feasible immobilization strategy, if low immobilization levels ≤ 300 µRIU are sufficient. In this case, ligand concentration and immobilization time should be significantly increased (≥0.5 mg/mL DBCO-ligand concentration, ≥30 min ligand incubation time).

Avoid prolonged incubation of the sensor chip in water, as this can negatively affect the integrity of the sensor coating over time. Instead, use a physiological buffer for storage.

Regeneration

The selection of a suitable regeneration buffer is crucial when performing binding studies in which the analyte does not dissociate completely within an adequate period of time. In such cases, the analyte must be removed manually through a regeneration procedure. The goal is to ensure complete analyte removal without reducing ligand activity. Since the specific binding between the ligand and analyte is driven by a unique – and, in most cases, unknown – combination of physical forces, the regeneration conditions must be determined empirically.

Experience has shown that short pulses of 10–20 mM H3PO4 or 10 mM Glycine-HCl at pH 1.5–2.5 (part of Regeneration Scouting Kit 1, product code K RK1-50I) are often sufficient to achieve quantitative regeneration. However, some receptor-ligand pairs may require different conditions for successful regeneration. Occasionally, the interaction between two binding partners is so strong that binding becomes practically irreversible. In such cases, kinetic titration or capture immobilization of the ligand are promising strategies.

Andersson has proposed an innovative algorithm to streamline the otherwise time-consuming process of identifying optimal regeneration conditions [3][4]. His approach involves systematically combining six different regeneration cocktails. The composition of these cocktails, which Xantec distributes as the Regeneration Scouting Kit 2 (product code K RK2-50I), is outlined in the table below:

Stock solution Product code Composition
Acidic B RCA-50ML 37.5 mM Oxalic acid, 37.5 mM H3PO4, 37.5 mM formic acid, 37.5 mM malonic acid pH 5.0
Alkaline B RCB-50ML 0.2 M Ethanolamine, 0.2 M Na3PO4, 0.2 M Piperazine, 0.2 M Glycine pH 9.0
Chaotropic B RCI-50ML 0.46 M KSCN, 1.83 M MgCl2, 0.92 M Urea, 1.83 M Guanidine·HCl
Non-polar, water-soluble B RCS-50ML 20 % (v/v) DMSO, 20 % (v/v) formamide, 20 % (v/v) ethanol, 20 % (v/v) acetonitrile, 20 % (v/v) 1-butanol
Detergent B RCD-50ML 0.3 % (w/v) CHAPS, 0.3 % (w/v) Zwittergent 3–12, 0.3 % (v/v) Tween 80, 0.3 % (v/v) Tween 20, 0.3 % (v/v) Triton X-100
Chelating B RCC-50ML 0.02 M disodium EDTA
Procedure for regeneration screening and optimization
1 Prepare the first regeneration cocktail by mixing one part of one of the six stock solutions with two parts of water.
2 Inject the analyte until equilibrium is reached.
3 Screening: Inject the first regeneration cocktail and measure its effect as a percentage of the analyte removed from the sensor chip. No effect corresponds to 0 % regeneration, while complete removal equals 100 % regeneration. If the regeneration efficiency is below 10 %, proceed to inject the next regeneration cocktail (1 part stock solution, 2 parts water). If the analyte level drops below 67 % of the original value, inject new analyte and re-saturate the surface. Repeat until all cocktails have been evaluated.
The regeneration efficacy Re is calculated by the following formula:
Re= (analyte loss)/(analyte level) × 100 %
4 Optimization: Identify the two to three regeneration cocktails with the highest Re and recombine them using a 2D (two best regeneration cocktails) or 3D (three best regeneration cocktails) experimental mixture design. Add water if the new cocktails do not reach 100 % volume. Re-evaluate the Re of the new regeneration solutions.
5 If regeneration remains insufficient, follow the trends observed in the previous optimization experiment and iterate until regeneration is satisfactory.
Note: Repeated short pulses of the regeneration solution are generally more effective than increasing the injection time.

Storage of used sensor chips

For later reuse, sensor chips can be stored either dry or wet under physiological conditions. When handling the sensor chip, avoid touching the top coating with gloves or tweezers.

Biacore users only: To prevent detachment of the glass chip in the instrument after chips have been stored under buffer or at 100 % humidity, we strongly recommend checking the mechanical stability of the assembly before inserting the chip cartridge into the instrument.

Reichert users only: If the sensor chip is intended for later reuse, use the refractive index matching foil instead of immersion oil when installing the sensor chip for the first time. Oil traces may contaminate the hydrogel top coating after chip removal, potentially causing irreversible damage to the immobilized ligand.

Dry storage
1 Dismount the used sensor chip from your SPR instrument.
2 Rinse the hydrogel surface of the sensor chip carefully with ultrapure water.
3 Optional: Carefully remove excess water from the edge of the hydrogel coating using a pipette. Place a droplet (30 µL for SCB and SCBS) of XanTec stabilization buffer onto the wet chip surface and allow it to spread, ensuring it covers the entire surface. Let it dry for approximately 60 minutes in a desiccator with desiccant (4A molecular sieve). This step helps prevent denaturation of the immobilized ligand and prolongs the shelf-life of the sensor chip.
4 Dry the sensor chip with a jet of filtered air or nitrogen.
5 Store the sensor chip dry, using a 3A or 4A molecular sieve, in a cold environment (-25 °C) under an inert gas atmosphere in a tightly sealed container. The stability of the sensor chip depends on the stability of the immobilized ligand. The underlying hydrogel coating should remain stable for several weeks to months.
6 Reinstallation
No protective top coating: Equilibrate the sensor chip to room temperature before opening the storage container, then insert the chip according to the instrument manufacturer‘s instructions.
Protective top coating applied: Equilibrate the sensor chip to room temperature before opening the storage container. Immerse the chip in physiological buffer for 10 minutes to remove the protective layer from the hydrogel coating. Rinse gently with ultra-pure water and carefully dry it using a stream of filtered air or nitrogen.
Wet storage
1 Dismount the used sensor chip from your SPR instrument.
2 Rinse the hydrogel surface of the sensor chip carefully with ultra-pure water. Place the sensor chip in a container filled with sterile filtered, physiological buffer and seal it tightly. For Cytiva sensor chips, 50 mL centrifugation tubes are applicable. Store the sensor chip refrigerated at 2–8 °C.
The stability of the sensor coating mainly depends on the stability of the immobilized ligand. The underlying hydrogel coating should be stable for several days to weeks at such conditions. Long-term storage in water is not advised, as this can negatively affect the integrity of the sensor coating.
3 Reinstallation
Remove the sensor chip from the container, preferably using clean tweezers. Rinse with ultra-pure water to remove buffer salts, and carefully dry it using a stream of filtered air or nitrogen. Then, insert the chip according to the instrument manufacturer‘s instructions.

Troubleshooting

Issue Possible solution
Insufficient electrostatic preconcentration Perform electrostatic preconcentration screening to check for optimal preconcentration conditions.
Desalt protein directly into Coupling buffer to remove possible salt contaminants. Lower the ionic strength of the Coupling buffer.
The ligand is too acidic (pI < 3.0) and does not preconcentrate on polycarboxylate sensor chips. In this case, alternative coupling methods should be considered.
Decrease the ionic strength of the immobilization buffer.
Insufficient protein immobilization level Make sure that you employ optimal electrostatic preconcentration conditions.
Make sure that all interfering components of your protein stock solution (such as free DBCO) are completely removed from your solution. Sometimes, multiple desalting steps are necessary for quantitative removal.
Make sure that DBCO has been successfully conjugated to your ligand. Increase the protein contact time.
Increase the protein concentration.
Insufficient ligand activity Check ligand integrity in your stock solution and in the immobilization buffer with regard to activity, aggregation, and biological contamination. Not all proteins tolerate low pH or low ionic strength.
Decrease the degree of labelling, as over-conjugated proteins with multiple DBCO-groups tend to crosslink inside the sensor matrix which results in overall low chip performance.
Decrease the overall immobilization level to minimize ligand crowding.
If your protein is sensitive to acidic pH, increase the pH of your Coupling buffer. If physiological conditions are required, increase protein concentration and contact times.
Sometimes, the ligand couples at its active site. In this case, alternative coupling methods should be considered.

Literature

  1. Dommerholt, J., Rutjes, F.P.J.T. and van Delft, F.L. 2016. Strain-Promoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides. Top Curr Chem (Z) 374: 16. DOI: https://doi.org/10.1007/s41061-016-0016-4
  2. Pickens C. J., Johnson S. N., Pressnall M. M., Leon M. A. and Berkland C. J. 2018. Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide–Alkyne Cycloaddition. Bioconjugate Chemistry 29 (3), 686–701. DOI: 10.1021/acs.bioconjchem.7b00633
  3. Andersson, K., Areskoug, D., & Hardenborg, E. (1999). Exploring buffer space for molecular interactions. Journal of Molecular recognition, 12(5), 310-315.
  4. Andersson, K., Hämäläinen, M., & Malmqvist, M. (1999). Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach. Analytical chemistry, 71(13), 2475-2481.

V. 10/24a

For in-vitro use only. Not for use in clinical diagnostic procedures.