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Instructions
for use

HCX Sensor Chips (PDF download)

Product description

Introduction

XanTec’s HCX sensor chips are based on a 3D hydrogel matrix composed of flexible polycarboxylate chains grafted onto a hydrophilic adhesion promoter on a gold support. The carboxylate groups of this coating are partially activated with NHS-esters in medium to high densities, typically resulting in high ligand immobilization densities. The pre-activation of HCX sensor chips significantly facilitates and accelerates ligand immobilization, providing a valuable tool to improve the experimental workflow. This is particularly advantageous for spot-immobilization on imaging SPR systems.

Though a certain fraction of the spotted ligand will immobilize when drying the spots, electrostatic preconcentration will significantly increase the immobilization yield. Therefore, a preconcentration scouting of the ligand (see protocol below) should be conducted before the actual immobilization. Small molecules can freely diffuse into the sensor matrix and do not require electrostatic preconcentration conditions for efficient immobilization.

Note: Preconcentration scouting must be conducted using a separate, non-activated HC sensor chip. HCX sensor chips are incompatible with the standard electrostatic preconcentration scouting typically performed prior to covalent immobilization.

Additional materials required for amine coupling

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

Ethanolamine quenching buffer (product code B EA85-50ML): 1 M ethanolamine*HCl, pH 8.5, 50 mL

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

Corresponding HC sensor chip¹ (optional) for electrostatic preconcentration scouting.

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

¹ If you intend to use a HCX200M sensor chip for ligand immobilization, perform the electrostatic preconcentration scouting with a corresponding HC200M sensor chip.

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. As the HCX are very vulnerable to hydrolysis, it is recommended to use the sensor chips directly after opening the pouch.

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 10–100 µg/mL are usually sufficient for efficient covalent coupling.

If the pI of your protein is unknown, you may want to perform an electrostatic preconcentration scouting. In this case, dilute your 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.

Note: Using HCX coatings, preconcentration scouting must be performed on a separate, non-activated HC sensor chip. It is not recommended to quench an abundant channel of an HCX sensor chip to scout for electrostatic preconcentration conditions, as this will leave the other channels prone to hydrolytic decay, ultimately leading to immobilization failure.

Protocol for amine coupling on HCX sensor chips

Notes

NHS ester of pre-activated HCX sensor chips hydrolyze quickly under neutral and alkaline pH-conditions. Therefore, it is not recommended to precondition the surface with Borate elution buffer but to start with the injection or spotting of the ligand in a suitable coupling buffer after 1–2 min of equilibration of the sensor chip with water (dry afterwards if ligand is spotted).

Physiological running buffer is not recommended as alternative running buffer as hydrolysis of NHS-esters progresses quickly at pH 7.4.

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

After immobilization, 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 [1][2]. 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:

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.

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.

Troubleshooting

Literature

1. Andersson, K., Areskoug, D., & Hardenborg, E. (1999). Exploring buffer space for molecular interactions. Journal of Molecular recognition, 12(5), 310-315.
2. 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.