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Instructions
for use

ZC Sensor Chips (PDF download)

Product description

Product code Prefix (designates the instrument): SCB, SCBS, SCBN, SPP, SCBI, SPSM, SCH, SPMX, SCR, SCS, SD +
Add: ZC30M, ZC80M, ZC150D
Example: SCBS ZC80M
Intended purpose ZC sensor chips are designed for use with EDC/NHS chemistry to enable covalent ligand immobilization. They are coated with a zwitterionic polymer that has an approximately equimolar ratio of carboxylic acids and tertiary amines. The balanced charge distribution on the chip surface minimizes nonspecific binding of both negatively and positively charged biomolecules, while also enabling the electrostatic preconcentration of highly acidic biomolecules. ZC sensor chips are particularly suited for situations where the ligand is difficult to immobilize on standard polycarboxylate sensor coatings, or when highly nonspecific analyte interactions with the sensor matrix pose an extraordinary challenge. For successful immobilization, the ligand must contain at least one reactive primary amino group. Recommended applications include the investigation of biomolecular interactions involving protein, 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
  • Amine coupling kit AZ, zwitterionic surfaces, product code K AZ-30I
  • ZCC sensor chip

Introduction

ZC sensor chips are coated with a zwitterionic polymer containing an almost equimolar ratio of carboxylic acids and tertiary amines. The ZC chip surface allows electrostatic pre-concentration and covalent coupling of even highly acidic ligands like DNA- and RNA-oligos under pH-neutral conditions, as long as at least one NHS-reactive amine is available for coupling. Ligands are immobilized by standard EDC/NHS-coupling following the procedure described below. In physiological buffers the balanced charge renders this chip coating inert against nonspecific binding of both, negatively and positively charged biomolecules. Therefore, the main area of application of ZC sensor chips are ligands that are difficult to immobilize due to a highly acidic character as well as analytes with multiple positive charges that would otherwise absorb strongly on classic polycarboxylate sensor chips.

The immobilization protocol is based on the standard procedure for EDC/NHS-coupling. Due to a near neutral pH of the recommended immobilization buffer and a high active ester (NHS) density, immobilization generally proceeds fast and efficient. Additionally, two different quenching buffers are available to fine-tune the charge of the resulting chip surface. Quenching with Quenching buffer A is ideal for achieving a high level of general bioinertness and an approximately neutral net surface charge. In contrast, quenching with Quenching buffer C yields a slightly positive net charge at physiological pH, which can be beneficial when working with highly positively charged analytes.1

ZC Sensor Chips
Fig. 1: Principle of ligand immobilization on ZC sensor chips: In the unactivated state, ZC sensor chips have a nearly neutral net charge, with approximately equal ratios of negative carboxyl and positive amine groups. 1. EDC/NHS activation converts a fraction of the carboxyl groups into neutral NHS esters, shifting the surface charge from neutral to positive. 2. The positively charged sensor surface can temporarily preconcentrate negatively charged ligands. 3. The ligand preconcentrates and covalently binds within the ZC sensor matrix. 4. Remaining NHS esters are quenched, returning the sensor chip to a neutral charge again.

Sufficient electrostatic preconcentration is a prerequisite for successful protein immobilization, so a preconcentration scouting of the ligand (see protocol below) should be performed before actual immobilization. However, electrostatic preconcentration is not possible on ZC sensor chips without activation. Therefore, one channel of the sensor chip must be derivatized with Quenching Buffer C to allow for electrostatic preconcentration scouting. Alternatively, electrostatic preconcentration scouting can be conducted on a separate ZCC sensor chip. This chip cannot be used for covalent coupling afterward but has charge conditions similar to an activated ZC sensor chip. ZCC scouting chips can be reused multiple times. Note that the pH of the coupling buffer should be above the ligand’s isoelectric point to ensure a negative net charge on the ligand, which is necessary for electrostatic preconcentration on the positively charged ZC chip surface.

1 Quenching with ethanolamine buffer is also feasible, resulting in a final net surface charge that falls between those of ZC sensor chips treated with Quenching buffers A and C.

Additional materials required for Amine Coupling

Amine coupling kit AZ, zwitterionic surfaces (product code K AZ-30I)

Optional: ZCC sensor chip (for electrostatic preconcentration scouting)

Ligand bearing reactive amino groups (to be provided by the user)

Notes on EDC

Even high-purity EDC·HCl often contains impurities such as diamines, which can neutralize negative surface charges and even quench the (sulfo-)NHS esters on the activated chip surface. Such artifacts can significantly decrease the immobilization yield, especially at high EDC concentrations, and in the worst-case scenario, the deactivation can be quantitative. Other components in the activation mixture may also introduce trace contaminants that interfere with the process. The EDC·HCl provided by XanTec has undergone purification and testing to confirm its suitability for SPR applications.

Preparations for Amine Coupling

Clean the SPR fluidics

Make sure that the flow system of your SPR equipment is free from any protein contamination, because even minor amounts of desorbed protein will concentrate onto the charged sensor surface. If necessary, clean the system with either 1 % Tween 20 or – more stringent – 0.5 % SDS for 5 min followed by 50 mM glycine*HCl (pH 9.5) for 10 min (both part of the Desorb Kit, product code K D-500ML). The glycine is necessary to remove traces of SDS.

Ligand preparation

In some cases, the ligand solution contains sodium azide or amine-containing buffers such as Tris. Both components can react with NHS esters and negatively affect the immobilization efficiency. Therefore, they should be removed from the ligand solution by a desalting procedure before immobilization. Generally, it is advisable to desalt into an azide- and amine-free, pH-neutral buffer with low ionic strength. Desalting directly into preconcentration buffer can increase the preconcentration effect after dilution but sometimes results in protein loss due to aggregation.

Use concentrated ligand stock solutions (≥1 mg/mL). Thereby, the final ligand solution in Coupling buffer is less affected by the pH and ionic strength of the stock solution.

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.

EDC preparation

Aqueous EDC solutions are susceptible to hydrolysis. Therefore, small aliquots of Activation Buffer should be frozen, and the corresponding amount of solid EDC·HCl should be dissolved immediately in the buffer before use. Because EDC is sensitive to humidity, the reagent bottle should be stored at -20 °C or lower in a desiccated container, brought to room temperature before opening, and exposed to air for as short a time as possible, ideally under an inert gas atmosphere.

Alternatively, solid EDC·HCl can be dissolved in ultra-pure water to prepare a 1 M EDC solution, which can then be divided into separate aliquots for storage at -20 °C or lower. These aliquots remain stable for up to two months when kept frozen. Immediately before use, thaw a frozen aliquot to room temperature, gently shake it, and combine it with the defrosted Activation buffer. This mixture can then be used for activating the ZC sensor chip.

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 above the pI of the ligand is recommended for efficient immobilization. Protein concentrations of 10–100 µg/mL are usually sufficient for efficient covalent coupling.

Due to its almost equimolar ratio of positively and negatively charged groups, the ZC sensor surfaces do not provide the option of an electrostatic pre-concentration from the start. This problem can be solved by using a ZCC sensor coating or by in-situ generation of a positively charged sensor surface in an abundant flow channel of your SPR-instrument, following the procedure below. If a ZCC sensor chip is used, skip steps 3–6.

Procedure for electrostatic preconcentration scouting 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 Abundant channel only: Mix solid EDC (0.5 M final EDC concentration) and Activation buffer. Filter EDC/Activation buffer solution (0.45-µm) and inject. 15 600
4 Wash with running buffer to remove occasional air bubbles. 200 30
5 Inject Quenching buffer C. Wait until the baseline has stabilized. 8 1200
6 Inject Borate elution buffer. Wait until the baseline has stabilized. 25 3 × 60
7 Inject your protein (5–25 µg/mL) in MES Coupling buffer pH 6.5. If no interaction occurs, the ionic strength or pH may be adjusted or the protein concentration slightly increased. Repeat the last two steps until a sufficient preconcentration of the protein is observed on the sensor surface. 10 300
8 Inject Borate elution buffer. Wait until the baseline has stabilized. 25 1-3 × 60

Notes

Never use sulfo-NHS in combination with ZC sensor chips, as no preconcentration of proteins will then be possible.

Electrostatic preconcentration on ZC chips becomes inefficient at pH 8.0 or higher. The recommended pH range for the electrostatic preconcentration of ZC sensor chips is pH 5.0–7.5.

Protocol for Amine Coupling

Procedure Flowrate
[µL/min]		 Injection
time [s]
1 Equilibrate your SPR-system with water as running buffer and mount a compatible XanTec ZC sensor chip.
2 Condition the surface with Borate elution buffer. Wait until the baseline has stabilized. 25 3 × 60
3 All channels: Mix solid EDC (0.5 M final EDC concentration) and Activation buffer. Filter EDC/Activation buffer solution (0.45-µm) and inject. 15 600
4 Optional: Wash with running buffer to remove occasional air bubbles. 200 30
5 Ligand channel only: Inject ligand solution in MES pH 6.5 (standard) or another suitable Coupling buffer. Protein concentrations of 10–100 µg/mL are recommended. 15 600
6 All channels: Inject Quenching buffer A or Quenching buffer C. Use Quenching buffer A (standard) for a neutral sensor charge or Quenching buffer C for a slightly positively charged surface. 10 900
7 Optional: remove loosely physisorbed protein with Borate elution buffer. 25 3 × 60
8 Switch to physiological running buffer and wait until the SPR-signal has stabilized.
9 Start interaction analysis. We recommend beginning with 3–5 consecutive regeneration cycles to improve data quality and stabilize the chip surface.

Notes

Physiological running buffer can be used instead of water during immobilization, but dispersion effects of protein coupling solution and running buffer can lower immobilization yields somewhat.

Avoid amine- or azide- containing buffers.

Higher immobilization yields can be achieved by lowering the ionic strength of the protein Coupling buffer, changing the pH of the protein solution, increasing the protein concentration and extending the ligand contact time.

If the SPR instrument does not support direct mixing of solid EDC with the Activation buffer prior to injection, EDC can be pre-dissolved at 1 M in ultra-pure water. Immediately before injection, this stock solution should be mixed 1:1 with the Activation buffer to yield a 0.5 M EDC working solution. The EDC–water stock remains stable for typical immobilization durations of 1–2 hours.

This protocol is also applicable for the efficient covalent immobilization of amino-functionalized oligos.

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 ultra-pure 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 scouting 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 alkaline (pI > 8.0) and does not preconcentrate on activated ZC sensor chips. In this case, alternative coupling methods should be considered.
  • If you observe an unusual high signal increase after EDC/NHS-activation, impure EDC might be the reason. Replace the EDC by a fresh vial or use a different brand.
  • 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 azide- or amine-containing buffers like Tris) are completely removed from your solution. Sometimes, multiple desalting steps are necessary for sufficient removal.
  • Increase the ligand contact time.
  • Increase the ligand 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 ionic strength.
  • Decrease the immobilization level by reducing the EDC concentration.
  • Use physiological pH values for your coupling buffer.
  • Sometimes, the ligand couples at its binding site. In this case, alternative coupling methods should be considered.

Literature

  1. Risse, Gedig, Gutmann: Carbodiimide-Mediated Immobilization of Acidic Biomolecules on Reversed-Charge Zwitterionic Sensor Chip Surfaces, Anal Bioanal. Chem. (2018) 410(17), 4109–4122.
  2. Bieri, M., Hendrickx, R., Bauer, M., Yu, B., Jetzer, T., Dreier, B., ... & Hemmi, S. (2021). The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and-3 infection. PLoS pathogens, 17(12), e1010083.
  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. 04/25a

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