NHS Surfaces

for coupling via the N-terminus of biochemical species

The NHS-ester reacts immediately with the NH2-terminus of biochemical species to form a covalent bond with the surface (420 kJ/mol). The reaction of carboxyl functionalities with N-hydroxysuccinimide leads to highly reactive esters, which can be easily reacted with nucleophiles e.g. amines or hydrazines. However, due to its high reactivity the NHS-ester is susceptible to hydrolysis and is characterized by a relatively short shelf-life. All NHS-activated surfaces should therefore be processed quickly.

There are a number of different approaches to couple on the NHS-surface:

  1. It is assumed that not all Carboxy groups have reacted to NHS-esters during activation. Thus, a negatively charged carboxy surface still remains, which supports the physico-chemical adsorption of positively charged probes e.g. NH3+. Hence, a protonating media (pH < 5) for the biochemical species getting a positively charge is required.
  2. A nucleophilic attack on the active ester is also catalyzed under basic conditions (pH > 8,5).

After attachment of the biochemical species the surfaces must be blocked with a blocking buffer containing small molecules that can access all reactive groups within the 3D-Matrix.

PolyAn equips glass slides, coverslips and polymer slides as well as 96-well plates with 3D-NHS surfaces. Please do not hesitate to contact us, if you would like to functionalize a different format or substrate with our 3D-NHS surface.

Selected Publications

NHS surface for the immobilization of Glycans:

  • Swan, J. et al., `The sialome of the retina, alteration in age-related macular degeneration (AMD) pathology and potential impacts on Complement Factor H´, bioRxiv, 2025, DOI: 10.1101/2025.03.09.642149.
  • Khan, N. et al., `Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits´, J. Biol. Chem., 2022, 298, 101900. DOI: 10.1016/j.jbc.2022.101900.
  • Sasmal, A. et al., `Simple and practical sialoglycan encoding system reveals vast diversity in nature and identifies a universal sialoglycan-recognizing probe derived from AB5 toxin B subunits´, Glycobiology, 2022, 32, 1101. DOI: 10.1093/glycob/cwac057.
  • Ji, Y. et al., `Reversible O‑Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition´, Chem. Biol., 2021, 16, 1951. DOI: 10.1021/acschembio.0c00998.
  • Saha, S. et al., `Exploring the Impact of Ketodeoxynonulosonic Acid in Host-Pathogen Interactions Using Uptake and Surface Display by Nontypeable Haemophilus influenzae´, mBio, 2021, 12, 03226-20. DOI: 10.1128/mBio.03226-20.
  • Siddiqui, S.S. et al., `Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis´, PNAS, 2021, 118, 2018090118. DOI: 10.1073/pnas.2018090118.
  • Ruprecht, C. et al., `Practical considerations for printing high-density glycan microarrays to study weak carbohydrate-protein interactions´, Carbohydrate Res., 2019, 481, 31. DOI: 10.1016/j.carres.2019.06.006.

NHS surface for the immobilization of Oligonucleotides (DNA/RNA):

  • Jin, Z. et al., `Cross-amplified Barcodes on Slides for Spatial Transcriptomics Sequencing´, bioRxiv, 2022, DOI: 10.1101/2022.08.25.504658.

NHS surface for the immobilization of Proteins and Antibodies:

  • Vendrell-Fernández, S. et al., `Conversion of the OmpF Porin into a Device to Gather Amyloids on the E. coli Outer Membrane´, ACS Synth. Biol., 2022, 11, 655. DOI: 10.1021/acssynbio.1c00347.

NHS surface for the immobilization of Peptides:

  • Rapsch, K. et al., `Identification of antimicrobial peptides and immobilization strategy suitable for a covalent surface coating with biocompatible properties´, Bioconjugate Chem., 2014, 25, 308. DOI: 10.1021/bc4004469.