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

Abstract

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.

Keywords: Prussian Blue; Western blotting; dot blot; immunoassay; immunohistochemistry; peroxidase.

Figure 1

Properties of PB@Gel/Str nanoparticles: (a) Absorbance spectrum. (b) Size of PB@Gel/Str in water, phosphate buffer, and Na-citrate buffers (pH 4–7). (c) Zeta potential at pH 4–7. (d) Catalytic activity with TMB substrate (1-PB@Gel/Str + TMB + H₂O₂; 2-PB@Gel/Str + TMB; 3-TMB + H₂O₂). (e) Catalytic activity with DAB substrate (1-PB@Gel/Str + DAB + H₂O₂; 2-PB@Gel/Str + DAB; 3-DAB + H₂O₂). (f) Binding of Bi-BSA (measured via an ELISA-like assay). (g) Storage stability of PB@Gel/Str and PB@Gel/BSA. (h) Binding of Bi-BSA (measured via a dot blot assay) (1-PB@Gel/Str; 2-PB@Gel/BSA). (i,j) TEM images of PB@Gel/Str. Mean ± SD, n = 3.

Figure 2

DAB oxidation by PB@Gel/Str and HRP. (a) Color of products generated by PB@Gel/Str and HRP. (b,c) Absorbance of reaction products at 470 and 800 nm. The molarity of all buffers was 33 mM. Nine parts of MES, HEPES, and TRIS–HCl buffers were mixed with one part of Na-citrate buffer with the same pH prior to the experiment. The concentration of H₂O₂ was 0.1% for both PB@Gel/Str and HRP. Mean ± SD, n = 4.

Figure 3

The effect of NaCl concentration on the intensity of DAB oxidation. (a) Absorbance of reaction products at 470 nm. (b) Absorbance of reaction products at 800 nm. The final concentrations of NaCl in the substrate are given. The molarity of all buffers was 33 mM. Nine parts of MES, HEPES, and Tris–HCl buffers were mixed with one part of Na-citrate buffer with the same pH prior to the experiment. Mean ± SD, n = 2.

Figure 4

The effect of metal cations on the intensity of DAB oxidation. (a,b) Absorbance of substrate at 470 and 800 nm after the addition of PB@Gel/Str. (c,d) Absorbance of substrate at 470 and 800 nm before the addition of PB@Gel/Str. Mean ± SD, n = 2. The final concentration of Me^2+/3+ in the substrate was 0.8 mM. The molarity of all buffers was 33 mM. Nine parts of MES, HEPES, and Tris–HCl buffers were mixed with one part of Na-citrate buffer with the same pH prior to the experiment.

Figure 5

The effect of imidazole on the intensity of DAB oxidation. (a) Absorbance of reaction products at 470 nm. (b) Absorbance of reaction products at 800 nm. The final concentration of imidazole was 10 mM. Before being added to the buffer, the pH of the imidazole solution was adjusted using HCl to match the pH of the buffer. The molarity of all buffers was 33 mM. Nine parts of MES, HEPES, and Tris–HCl buffers were mixed with one part of Na-citrate buffer with the same pH prior to the experiment. Mean ± SD, n = 2.

Figure 6

Comparison of optimized DAB substrates with commercial formulations via dot blot assay (a,b) and in 96-well plates (c,d). (a) Location of Bi-BSA spots in the wells. (b) Direct Bi-BSA detection by PB@Gel/Str in white polystyrene plates. (c) Absorbance of substrates at 470 and 800 nm after the addition of PB@Gel/Str. (d) Absorbance of substrates at 470 and 800 nm in the absence of nanozymes. Substrates for (b–d): C1–200 mM Na-acetate buffer with 0.54 mg/mL DAB, 1 mM DTPA, 50 mM imidazole, and 0.03% H₂O₂, pH 5; C2–200 mM Na-acetate buffer with 0.54 mg/mL DAB, 1 mM DTPA, 50 mM imidazole, and 0.1% H₂O₂, pH 5; 3–33 mM Na-citrate buffer with 1.5 M NaCl, pH 6, 1 mg/mL DAB, 0.1% H₂O₂; 4–33 mM Na-citrate buffer with 1.5 M NH₄Cl, pH 7, 1 mg/mL DAB, 0.1% H₂O₂; 5–30 mM HEPES–HCl buffer with 3 mM Na-citrate and 1.5 M NaCl, pH 7, 1 mg/mL DAB, 0.1% H₂O₂. Mean ± SD, n = 3.

Figure 7

Immunohistochemical staining of pancreatic islets with horseradish peroxidase (a,d) and Prussian Blue nanoparticles (b,e–g). (c) Data on staining intensity obtained after digital processing of histological images taken with different DAB solutions (BG—background). Commercial DAB solution (a–d) or 100 mM Na-citrate buffer with 1 mg/mL DAB, 1 and 0.1% H₂O₂, pH 3 (e), pH 5 (f), or pH 6 (g) were used for staining.

Figure 8

(a–c) Dot blot assay of IgG against SARS-CoV-2 spike protein (two spots of spike protein) in pooled human sera obtained from seronegative (−) individuals (dilution 1:100) and seropositive (+) individuals (dilution 1:100 and 1:1000). Bi-BSA and hIgG were used as positive controls, while BSA served as the negative control (one spot for each). (a) Schemes of the assay are provided for each spot. (b) Test strips. (c) Quantitative analysis of spot intensity. (d) SDS-PAGE of BSA, Bi-BSA, IgG from human serum (hIgG), and mouse monoclonal IgG2a (mIgG). Coomassie Brilliant blue staining. The molecular weight values of protein markers are given. (e) Western blotting of the same samples.