Afficoat™ for Reducing Non-Specific Adsorption in SPR Experiments
Updated: Oct 1, 2021
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 [1, 2].
Surface plasmon resonance (SPR) biosensor surfaces can also succumb to non-specific adsorption. The sensitivity of the response of the actual target biomarker can be affected by the localized increase in mass due to the non-specific adsorption of proteins, which would change the local refractive index . Dilution of biological samples is not ideal as the concentration of biomarkers is already quite low. The solution is to use a surface coating that will minimize the level of protein 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 (see Figure 1), and the carboxyl end of the peptide can be used to immobilize capture biomolecules.
Figure 1. Schematic diagram depicting how proteins or His-tagged proteins can be immobilized directly on Afficoat™ sensor chip via coupling agents or via a nitroloacetic acid (NTA) analog, respectively. X, Y, and Z represent different amino acids and the sequence shown is the pattern for Afficoat.
The focus of this blog is to present the performance of Afficoat compared to other SAMs of different peptide sequences and standard surface coatings at minimizing non-specific adsorption in an SPR experiment while maintaining the capture biomolecules' function. Furthermore, the use of Afficoat-modified sensor chips with Affinité's P4SPR™ instrument for various clinical applications will be summarized.
Determination of the Optimal Peptide Sequence
A study was conducted to determine the optimal peptide sequence that can best minimize non-specific adsorption. The SPR experiment was accomplished by setting up a dove prism SPR instrument in the Kretchmann configuration . After baseline stabilization, 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. The chips were then rinsed with PBS for 5 min. Then, the amount of adsorbed proteins was quantified. Table 1 lists the peptide sequence patterns that were used and the results are in Figure 2. As it turns out, sequence #5 demonstrated the least level of non-specific adsorption. Hence, this peptide sequence became what is now known as Afficoat™.
Table 1. List of peptide sequence that was investigated. 3-MPA stands for 3-mercaptopropionic acid. X, Y, and Z represent different amino acids.
Figure 2. Comparison of non-specific adsorption levels between different peptide sequences. The reported level of non-specific adsorption by PEG is marked by the yellow line .
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. For example, it has been shown that His-tagged hDHFR (human dihydrofolate reductase) remained active when it was immobilized using Afficoat coating and the subsequent addition of copper ions and an NTA analog (see Figure 1) . The activity of the enzyme was determined to be (4.4 +/- 0.8) x 10-5 U, which was equivalent to (5 +/- 0.9) ng (n=4) of the fully active enzyme in solution .
To demonstrate the retention of enzymatic activity of an immobilized enzyme on Afficoat, the equilibrium dissociation constant (KD) was determined for a His-tagged maltose binding protein-IgG system. The His-tagged fusion protein was immobilized as depicted in Figure 1. Increasing concentrations of IgG were flowed into the system and were left to react for 5 min each time. 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
To give some perspective, Figure 3 compares the non-specific adsorption levels between Afficoat and two other commonly used surface chemistries, PEG and CM-Dextran . Needless to say, Afficoat excels in minimizing non-specific adsorption compared to other surface chemistries under the same experimental conditions.
Figure 3. Comparison of non-specific adsorption levels when different surface coatings were exposed to bovine serum containing 76 mg/mL of proteins .
Further Applications of Afficoat™
Since Afficoat had shown promise in minimizing non-specific adsorption in the earlier set of experiments, it has been used to detect other proteins, hormones, and molecules in complex biological samples. Afficoat sensor chips have been used in the prototype of Affinité's P4SPR™ for the therapeutic drug monitoring of methotrexate, an anti-cancer and immunomodulatory drug, in human serum samples. The results agreed well with those obtained by LC-MS/MS and fluorescence polarization immunoassay . The detection of testosterone was achieved in a competitive assay on Afficoat-functionalized sensor chips . Most recently, the detection of antibodies specific for the SARS-CoV-2 virus in clinical samples such as serum, plasma, and dried blood spots was achieved by using the Afficoat-modified sensor chips in the P4SPR [7, 8]. Another large study involved serum samples from individuals who were COVID-19 positive to investigate the cross-reactivity between antibodies in those serum samples with native and variant SARS-CoV-2 spike proteins .
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 such as PEG and CM-Dextran. It also allows enzymes to retain their activities and 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.
The Affinité Advantage
Affinité Instruments’ P4SPR™ is a very user-friendly, compact, and portable instrument. In addition, samples do not need much preparation and can be manually injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.
Simplicity - Fast training, fast results
Versatility - Pharmaceutical, biosensing, assay development applications
Economy - Affordable, accessible
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