Low-cost colorimetric method for modeling protein-nanoparticle interactions

Quantitative characterization of nanoparticle (NP) and protein interactions using a high-throughput mode is a challenge. In an article published in the journal Analytical Chemistry, the researchers took a colorimetric approach to the quantitative analysis of proteins adsorbed on the surface of NP.


Study: High-performance colorimetric analysis of nanoparticle-protein interactions based on the enzymatic-mimetic properties of nanoparticles. Image credit: Angel Soler Gollonet / Shutterstock.com

Interactions between nanomaterials and proteins

The designed nanomaterials are applied in biomedical fields for bioimaging, disease diagnosis, and drug delivery. Upon entering a biological environment, nanomaterials adsorb serum proteins and instantly form a “protein crown” that can alter the physicochemical properties of nanomaterials. Thus, altered nanomaterials determine the immune response and biological behaviors of proteins such as targeting ability, cytotoxicity, and cell internalization.

Therefore, it is necessary to understand the interactions between nanomaterials and proteins to design effective and safe functionalized nanomaterials for clinical applications. Despite continuous efforts by researchers to understand nano-bio interactions, the behavior of protein adsorption on the surface of the nanomaterial is still unclear. To this end, the versatility in nanomaterials and the lack of efficient characterization tools could be attributed as possible reasons for the lack of clarity in nano-bio interactions.

Although biophysical techniques illustrate the mechanical aspects of protein-nanomaterial interactions, these techniques cannot achieve a high-performance quantitative analysis of the protein crown due to complex operating systems or the susceptibility of the crown. proteins to environmental degradation. This inefficiency in existing methods limits the extraction of important information about protein-NP interactions. Therefore, it is imperative to develop robust analytical tools for the quantitative characterization of protein-NP interactions in a high-performance manner.

Nanoparticles exhibit peroxidase-like intrinsic activity influenced by the surface environment. Therefore, it is a challenge for NPs to adsorb the analytes from the solution. This adsorption difficulty negatively affects the catalytic efficiency in colorimetric reactions. Consequently, the solution shows changes in absorbance values, reflecting adsorption events.

Cationic NPs can easily bind to biomolecules and undergo cell internalization. Therefore, they are widely applied in biomedicine. However, the study of cationic NP interactions with biological systems remains virtually unexplored.

High-performance colorimetric analysis of nanoparticle-protein interactions

In the present work, the researcher proposed a new colorimetric approach for the quantitative analysis of protein adsorption on the NP surface due to its peroxidase-like properties. They considered cationic gold (Au) NPs to demonstrate the ability of the colorimetric method to evaluate NP-protein interactions in multipole plates using a high-performance mode. The team obtained the binding parameters of three different serum proteins and human proteins to AuNPs. From the quantitative analysis of NP-protein interactions, they observed that the binding affinity and the consequent efficiency of inhibition of AuNP nanozyme activity were affected by the properties of the bound proteins and the size. of NP.

Research results

NP-protein interactions were evaluated based on colorimetric reactions that mimicked peroxidase. The researchers first studied the interactions of AuNPs whose average diameter was 4.6 nanometers (called AuNPs-5) with bovine serum albumin (BSA).

The results revealed the peroxidase-like catalytic properties of AuNPs-5 and its ability to oxidize the 3,3 ‘, 5,5’-tetramethylbenzidine (TMB) substrate in a blue product, the maximum absorbance of which is it was 652 nanometers in the presence of hydrogen. peroxide (H2O2). From the control experiment, the researchers confirmed the need for H2O2 and AuNPs for the oxidation of TMB and optimized the concentration of TMB and H2O2 to 4 millimoles and 1.2%, respectively, to produce the largest colorimetric changes.

The physiological conditions of the NP-protein interactions were simulated by performing colorimetric experiments in phosphate-buffered saline (PBS) with a pH of 7 and a temperature of 37 degrees Celsius. The gradual decrease in absorbance with increasing BSA concentration in the AuNPs solution suggested catalytic activity that mimics N, N, N-trimethylammonium bromide peroxidase (MUTAB) -AuNPs, the activity of which was weakened by the presence of BSA, thereby inhibiting the proximity of the substrate. on the surface of AuNP. The team observed that the characteristics of the protein crown, such as the composition of the crown and the orientation of the protein, play a role in the catalytic processes.

By adjusting the results to the modified Hill equation, they were able to obtain an agreement that gave a dissociation coefficient (KD ‘) of 3.5 ± 0.4 × 10−8 molar units. In addition, the maximum efficiency obtained (Emax) of 0.84 ± 0.03 confirmed the high suppressive effect of BSA on the peroxidase-like activity of AuNPs.


In conclusion, the researchers demonstrated that mechanistic studies of protein adsorption in NP with enzyme-like activity could be performed using a colorimetric platform. Cationic MUTAB-AuNPs with peroxidase-like activity were used to demonstrate that the colorimetric method can evaluate interactions between NP protein in multipole plates in a high-performance manner. Quantitative analysis of colorimetric data revealed that the characteristics of the protein strongly influenced the binding affinity and the consequent inhibitory activity of MUTAB-AuNP nanozymes. The inhibitory activity of this nanozyme mimicked natural enzymes in terms of protein binding to active sites, resulting in a significant loss of catalytic activity.


Mengyao Wen, Juanmin Li, Wencheng Zhong, Jie Xu, Shaohua Qu, Hui Wei and Li Shang (2022). High-performance colorimetric analysis of nanoparticle-protein interactions based on the enzymatic-mimetic properties of nanoparticles. Analytical Chemistry. https://pubs.acs.org/doi/10.1021/acs.analchem.2c01618

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