Prussian blue nanozymes possessing peroxidase-like activity gather significant attention as alternatives to natural enzymes in therapy, biosensing, and environmental remediation. Recently, Prussian blue nanoparticles with enhanced catalytic activity prepared by reduction of FeCl3/K3[Fe(CN)6] mixture have been reported. These nanoparticles were denoted as ‘artificial peroxidase’ nanozymes. Our study provides insights into the process of their synthesis. We studied how the size of nanozymes and synthesis yield can be controlled via adjustment of the synthesis conditions. Based on these results, we developed a reproducible and scalable method for the preparation of ‘artificial peroxidase’ with tunable sizes and enhanced catalytic activity. Nanozymes modified with gelatin shell and functionalized with affine molecules were applied as labels in colorimetric immunoassays of prostate-specific antigen and tetanus antibodies, enabling detection of these analytes in the range of clinically relevant concentrations. Protein coating provides excellent colloidal stability of nanozymes in physiological conditions and stability upon long-term storage.
Research paper on sunthesis of protein nanoparticles<div><br><div><b>Abstract</b></div><div>The desolvation
technique is one of the most popular methods for preparing protein
nanoparticles for medicine, biotechnology, and food applications. We fabricated
11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by
desolvation method. BSA nanoparticles from 2 batches were cross-linked by
heating at +70 °C for 2 h; other nanoparticles were stabilized by
glutaraldehyde. We compared several analytical approaches to measuring their
concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay,
and alkaline hydrolysis combined with UV spectroscopy. We revealed that the
cross-linking degree and method of cross-linking affect both Bradford and BCA
assay. Direct measurement of protein concentration in the suspension of purified
nanoparticles by dye-binding assays can lead to significant (up to 50-60%)
underestimation of nanoparticle concentration. Quantification of non-desolvated
protein (indirect method) is affected by the presence of small nanoparticles in
supernatants and can be inaccurate when the yield of desolvation is low. The
reaction of cross-linker with protein changes UV absorbance of the latter.
Therefore pure protein solution is an inappropriate calibrator when applying UV
spectroscopy for the determination of nanoparticle concentration. Our
recommendation is to determine the concentration of protein nanoparticles by at
least two different methods, including gravimetric analysis.<div><br></div></div></div>
Prussian blue nanozymes exhibit peroxidase-like catalytic activity and are therefore considered a stable and inexpensive alternative to natural peroxidases in the enzyme-linked immunosorbent assay (ELISA). In this work, we propose a robust method of Prussian blue nanozyme functionalization, which relies on the entrapment of nanozymes into albumin nanoparticles. The principle of the method is the addition of ethanol to a solution that contains albumin and nanozymes. At a high ethanol concentration solubility of albumin decreases, resulting in the formation of albumin nanoparticles loaded with nanozymes. The hydrodynamic diameter of nanoparticles was between 120 and 230 nm and depended on the nanozyme-to-BSA ratio. Encapsulation efficiency of nanozymes reached 96–99% and up to 190 μg of nanozymes were loaded per 1 mg of nanoparticles. Nanoparticles were stable at pH 5.5–7.5 and upon long-term storage in deionized water. Excellent reproducibility of the synthesis procedure was confirmed by the preparation of three individual batches of Prussian-blue-loaded BSA nanoparticles with almost identical properties. Nanoparticles were functionalized with monoclonal antibodies using glutaraldehyde cross-linking. The resulting conjugates were applied as labels in an ELISA-like assay of tumor marker prostate-specific antigen (PSA). The lower limit of detection was below 1 ng/mL, which enables measurement of PSA in the range of clinically relevant concentrations.
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