Non-specific adsorption is one of the most common sources of unreliable SPR data. Afficoat — Affinité's proprietary surface chemistry — addresses this problem at the molecular level.
Introduction
Selection of a surface chemistry for biosensing, screening, or assay development experiments requires careful consideration of several factors such as the type of linkage chemistry required and type of samples to be analyzed. If the target (bio)molecules exist in a complex biological sample, then a high level of protein adsorption is expected to occur on sensor surfaces. Non-specific adsorption can greatly impact the sensitivity of biosensor surfaces, and hence the quality of results. There can be as many as 1–10 mg/mL non-specific proteins in cell culture media, 30–60 mg/mL in crude cell lysate, and 40–80 mg/mL in serum.
SPR biosensor surfaces can also succumb to non-specific adsorption. The Affinité team has extensive experience in surface chemistry used in minimizing non-specific adsorption for SPR analysis. The culmination of these studies led to our proprietary surface coating reagent called Afficoat™. It is a self-assembled monolayer (SAM) composed of thiol-terminated peptides that are hydrophilic and zwitterionic. Its immobilization to gold sensor surfaces is achieved by thiol-gold chemistry, and the carboxyl end of the peptide can be used to immobilize capture biomolecules.
Determination of the Optimal Peptide Sequence
A study was conducted to determine the optimal peptide sequence that can best minimize non-specific adsorption. Gold sensor chips modified with different peptide SAMs were exposed to crude bovine serum with a protein concentration of 76 mg/mL for 20 min. After rinsing, the amount of adsorbed proteins was quantified. Sequence #5 demonstrated the least level of non-specific adsorption — and this peptide sequence became what is now known as Afficoat™.
Demonstration of Active Enzyme Activity and Determination of KD
Afficoat™ can serve as a linker to immobilize capture biomolecules that are His-tagged or amine-terminated. It has been shown that His-tagged hDHFR (human dihydrofolate reductase) remained active when immobilized using Afficoat coating. To demonstrate the retention of enzymatic activity, the equilibrium dissociation constant (KD) was determined for a His-tagged maltose binding protein-IgG system. The KD was determined to be (9.6 ± 0.3) nM, which agreed with results obtained by other techniques for antigen-IgG interactions.
Afficoat™ Outperforms Other Well-Known Surface Chemistries
Afficoat excels in minimizing non-specific adsorption compared to PEG and CM-Dextran under the same experimental conditions. When different surface coatings were exposed to bovine serum containing 76 mg/mL of proteins, Afficoat consistently showed the lowest level of non-specific adsorption.
Further Applications of Afficoat™
Since Afficoat had shown promise in minimizing non-specific adsorption, it has been used to detect other proteins, hormones, and molecules in complex biological samples. Applications include therapeutic drug monitoring of methotrexate in human serum, detection of testosterone in a competitive assay, and detection of antibodies specific for SARS-CoV-2 in clinical samples such as serum, plasma, and dried blood spots.
Conclusions
Afficoat™ is a SAM composed of a specific sequence of peptides that has been shown to minimize non-specific adsorption from proteins in real complex biological samples compared to other peptide sequences and commonly used surface chemistries. It also allows enzymes to retain their activities and enables determination of KDs of antigen-antibody interactions. Most importantly, it has shown tremendous potential for the detection of biomarkers, antibodies, and hormones in real clinical samples.