Optimizing the Composition of the Substrate Enhances the Performance of Peroxidase-like Nanozymes in Colorimetric Assays: A Case Study of Prussian Blue and 3,3'-Diaminobenzidine.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

Vertical Flow Immunoassay Based on Carbon Black Nanoparticles for the Detection of IgG against SARS-CoV-2 Spike Protein in Human Serum: Proof-of-Concept.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5-7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.

Prussian Blue Nanozymes with Enhanced Catalytic Activity: Size Tuning and Application in ELISA-like Immunoassay.

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.

Measuring the concentration of protein nanoparticles synthesized by desolvation method: Comparison of Bradford assay, BCA assay, hydrolysis/UV spectroscopy and gravimetric analysis.

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.

Nuclear magnetic resonance immunoassay of tetanus antibodies based on the displacement of magnetic nanoparticles.

A nuclear magnetic resonance (NMR) immunoassay based on the application of carbon-coated iron nanoparticles conjugated with recognition molecules was designed. The principle of the assay is that ELISA plates are coated with a capture element, and then an analyte is added and detected by conjugating the magnetic nanoparticles with recognition molecules. Afterwards, the elution solution (0.1-M sodium hydroxide) is added to displace the magnetic nanoparticles from the well surfaces into the solution. The detached magnetic nanoparticles reduce transverse relaxation time (T2) values of protons from the surrounding solution. A portable NMR relaxometer is used to measure the T2. Magnetic nanoparticles conjugated with streptavidin, monoclonal antibodies, and protein G were applied for the detection of biotinylated albumin, prostate-specific antigen, and IgG specific to tetanus toxoid (TT). The limit of detection of anti-TT IgG was 0.08-0.12 mIU/mL. The reproducibility of the assay was within the acceptable range (CV < 7.4%). The key novelty of the immunoassay is that the displacement of the nanoparticles from the solid support by the elution solution allows the advantages of the solid phase assay to be combined with the sensitive detection of the T2 changes in a volume of liquid.

Solid-phase nuclear magnetic resonance immunoassay for the prostate-specific antigen by using protein-coated magnetic nanoparticles.

A solid phase NMR-based sandwich immunoassay for the prostate-specific antigen (PSA) is presented. Carbon-encapsulated iron nanoparticles were functionalized with bovine serum albumin, coupled to monoclonal antibodies, and then used as magnetic labels. A nitrocellulose membrane with 8-µm pores was coated with capture antibodies and subsequently incubated with a serum sample and a suspension of the nanoconjugate. Test strips were placed in a portable homemade NMR relaxometer. Magnetic nanoparticles attached to nitrocellulose decrease the T2 relaxation time of the water protons located inside the pores of the membrane. Thus, T2 is inversely proportional to the concentration of the antigen (PSA) in the sample. The assay can be performed within 4 h. The detection limit is 0.44 ng mL-1. Kallikrein 2, human chorionic gonadotropin, and α-fetoprotein do not interfere. Graphical abstractSchematic representation of NMR relaxometry-based sandwich dot blot immunoassay of a prostate-specific antigen (PSA). Magnetic nanoparticles bound to immunosorbent decrease the transverse relaxation times (T2) of the water protons located within the pores of the membrane. RF coil: radiofrequency coil.

Magnetic Nanoclusters Coated with Albumin, Casein, and Gelatin: Size Tuning, Relaxivity, Stability, Protein Corona, and Application in Nuclear Magnetic Resonance Immunoassay.

The surface functionalization of magnetic nanoparticles improves their physicochemical properties and applicability in biomedicine. Natural polymers, including proteins, are prospective coatings capable of increasing the stability, biocompatibility, and transverse relaxivity (r2) of magnetic nanoparticles. In this work, we functionalized the nanoclusters of carbon-coated iron nanoparticles with four proteins: bovine serum albumin, casein, and gelatins A and B, and we conducted a comprehensive comparative study of their properties essential to applications in biosensing. First, we examined the influence of environmental parameters on the size of prepared nanoclusters and synthesized protein-coated nanoclusters with a tunable size. Second, we showed that protein coating does not significantly influence the r2 relaxivity of clustered nanoparticles; however, the uniform distribution of individual nanoparticles inside the protein coating facilitates increased relaxivity. Third, we demonstrated the applicability of the obtained nanoclusters in biosensing by the development of a nuclear-magnetic-resonance-based immunoassay for the quantification of antibodies against tetanus toxoid. Fourth, the protein coronas of nanoclusters were studied using SDS-PAGE and Bradford protein assay. Finally, we compared the colloidal stability at various pH values and ionic strengths and in relevant complex media (i.e., blood serum, plasma, milk, juice, beer, and red wine), as well as the heat stability, resistance to proteolytic digestion, and shelf-life of protein-coated nanoclusters.

Conjugation of carbon coated-iron nanoparticles with biomolecules for NMR-based assay.

In this work, we developed and optimized conjugates of carbon-coated iron nanoparticles (Fe@C) with streptavidin and monoclonal antibodies. The conjugation procedure included two stages. First, amino groups were grafted onto the carbon shell to facilitate noncovalent sorption of bovine serum albumin (BSA). Further, the covalent attachment of proteins to the BSA layer via glutaraldehyde coupling was performed. It was established and confirmed that the synthesis procedure is reproducible and allows preparation of stable conjugates. The resulting nanoparticles are clusters of Fe@C particles coated by proteins. The size of the clusters is in the range of 100-190 nm and can be controlled via the tuning of conjugation conditions, including pH, BSA-to-Fe@C ratio, etc. Conjugates of Fe@C with streptavidin and monoclonal antibodies (sizes of approximately 140-150 nm) were synthesized. Proton T2 relaxometry was used to detect these conjugates with very high sensitivity due to the magnetic markers, Fe@C. The relaxivity (r2) of different conjugates varied within the range of 290-450 1/s*mM. Conjugate applicability for relaxometry-based assay was confirmed by direct detection of streptococcal protein G and biotinylated BSA in a dot immunoassay.

Highly Stable Conjugates of Carbon Nanoparticles with DNA Aptamers.

Conjugates of carbon nanoparticles and aptamers have great potential in many areas of biomedicine. In order to be implemented in practice, such conjugates should keep their properties throughout long storage period in commonly available conditions. In this work, we prepared conjugates of carbon nanoparticles (CNP) with DNA aptamers using streptavidin-biotin reaction. Obtained conjugates possess superior stability and kept their physical-chemical and functional properties during 30 days at +4 °C and -20 °C. Proposed approach to conjugation allows loading of about 100-120 pM of biotinylated aptamer per 1 mg of streptavidin-coated CNP (CNP-Str). Aptamer-functionalized CNP-Str have zeta potential of -34 mV at pH 7, mean diameter of 168-177 nm, and polydispersity index of 0.080-0.140. High reproducibility of functionalization was confirmed by preparation of several batches of CNP-aptamer with the same size distribution and aptamer loading using independently synthesized parent CNP-Str nanoparticles. Stability of CNP-aptamer conjugates was significantly enhanced by postsynthesis addition of EDTA that prevents nuclease degradation of immobilized aptamers. Obtained nanoparticles were stable at pH ranging from 6 to 10. Optical properties of CNP-aptamer nanoparticles were also studied and their ability to quench fluorescence via Förster resonance energy transfer was shown. Taking into account properties of CNP-aptamer conjugates, we suppose they may be used in both homo- and heterogeneous colorimetric, fluorescent, and aggregation-based assays.