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.

3,3',5,5'-Tetramethylbenzidine (TMB) remains one of the most widely utilized chromogenic substrates for horseradish peroxidase (HRP) in colorimetric immunoassays, including enzyme-linked immunosorbent assays (ELISA). Despite its introduction into ELISA workflows over four decades ago, limited research has been conducted to systematically optimize TMB substrate formulations. Recent advancements in the field have proposed innovative approaches to enhance HRP catalysis, such as the use of deep eutectic solvents and ionic liquids, alongside investigations into the chemical properties of TMB and its analogs to identify more efficient alternatives. However, the development of stable and high-performance TMB solutions for clinical diagnostics requires a comprehensive understanding of how formulation parameters influence signal intensity and stability. In this study, we address these gaps by conducting a systematic evaluation of key factors affecting TMB substrate performance, including buffer pH, composition and molarity, specific ion effects, incorporation of organic solvents, and the use of polymer stabilizers. Additionally, novel strategies for signal amplification, identified through an extensive review of literature and patents, were experimentally tested. Based on these findings, we developed an optimized TMB formulation that was benchmarked against existing formulations reported in the literature.

Lanthanide complexes with organic ligands exhibit unique photophysical properties, including long-lived emission lifetimes, large Stokes shifts, and sharp emission bands, making them highly attractive for bioanalytical and biomedical applications. Incorporating these complexes into nanoparticles can further enhance signal intensity and assay sensitivity in both in vitro and in vivo settings. Here, we report the first successful use of the desolvation method to synthesize bovine serum albumin (BSA) nanoparticles loaded with europium complexes. This simple approach involved the dropwise addition of an ethanolic solution of europium complex precursors into an aqueous BSA solution, followed by thermal cross-linking to generate disulfide bonds. We systematically optimized synthesis parameters—including pH, precursor concentration, and addition rate—to produce nanoparticles with high brightness and low polydispersity. Under optimized conditions, the resulting nanoparticles exhibited a uniform size of 150–160 nm (PDI<0.15) as determined by dynamic light scattering and electron microscopy. Each nanoparticle encapsulated approximately 350 europium complexes, achieving quantum yields of 0.77–1.18% with excitation/emission maxima at 360/615 nm. The synthesis yield was ~80%, and the nanoparticles demonstrated excellent size stability across physiological pH conditions and after three months of storage. However, luminescence intensity decreased over time and under certain buffer conditions. Cytotoxicity studies using Vero cells showed cell viability above 80% at nanoparticle concentrations up to 1 mg/mL. The key challenges to be addressed are the improvement of synthesis reproducibility and the stability of luminescent properties.

One of the emerging trends in modern analytical and bioanalytical chemistry is the substitution of enzyme labels, such as horseradish peroxidase, with nanozymes — nanoparticles that possess enzyme-like catalytic activity. Since enzymes and nanozymes typically have different catalytic mechanisms, it is expected that the optimal reaction conditions will also differ. The objective of this paper was to optimize the diaminobenzidine (DAB) chromogenic substrate to enhance the performance of Prussian Blue nanozymes in immunostaining applications. We elucidated that enhancers (such as imidazole), which are typically added to commercial DAB substrate solutions to boost the color signal generated by peroxidase, can suppress the activity of nanozymes. This issue can be resolved by optimizing a nanozyme-specific substrate buffer composition. More specifically, we 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 provide a higher signal than commercial DAB formulations. Moreover, the composition of the substrate solution (type of buffer, ionic strength, additives) affects signal intensity to the same degree as buffer pH. Tuning the substrate buffer composition is, therefore, a simple and efficient way of signal amplification in nanozyme-based assays. Optimized DAB substrate formulations were applied to Prussian Blue-based tissue staining, western blotting assay of immunoglobulins, and dot blot assay of antibodies against SARS-CoV-2.

We report the first analytical application of albumin nanoparticles loaded with luminescent europium complexes for immunoassay development. These nanoparticles, synthesized via a desolvation method, exhibited a uniform spherical morphology with a hydrodynamic diameter of 263 nm and strong, long-lived luminescence at 615 nm (λex = 360 nm). Surface functionalization with streptavidin enabled specific binding to biotinylated proteins. The nanoparticles were applied as labels in a sandwich time-resolved solid-phase immunoassay for human IgG detection in black 96-well plates. Unlike commercial DELFIA assays, the method eliminates the need for signal enhancement steps, as the nanoparticles intrinsically contain high concentrations of europium complexes. Optimization studies revealed that the sharp emission peaks of europium can compromise assay reproducibility; however, employing surface scanning and increasing measurement replicates per well partially mitigated this effect. Time-resolved detection reduced background by two orders of magnitude and increased signal intensity nearly tenfold in IgG-positive samples. The assay demonstrated minimal cross-reactivity with IgA and IgM (~2%) and enabled IgG detection at serum dilutions up to 1:100,000. Comparative analysis showed strong concordance with commercial immunoassays and no concentration-dependent bias. The primary limitation observed was suboptimal intra-assay reproducibility (CV > 20% in four of six tested sera).