The use of traditional colored labels does not fully address the challenges of low sensitivity in rapid tests and insufficient stability of the reagents employed and necessitates additional procedural steps or complex instrumentation to generate an analytical signal. Owing to their unique properties, carbon black nanoparticles (CBNPs) present a promising solution to these issues. However, the current scientific data regarding the reproducibility, scalability, and preservation of structural and functional stability of CBNP-based colored labels remains insufficient for their widespread adoption in routine laboratory practice. Therefore, we have developed and optimized a reproducible method for the functionalization of oxidized CBNP with recognition elements, aimed at producing highly efficient vertical flow immunoassays (VFIAs). Our results demonstrate that the resulting diagnostic reagents maintain their structural and functional properties for one year when stored at +4 °C or −20 °C. We have demonstrated for the first time the reproducibility of the method for producing diagnostic reagents based on oxidized CBNPs with monoclonal antibodies. The novel VFIA based on oxidized CBNPs for the determination of C-reactive protein demonstrates a limit of detection of 2.6 ng/mL compared to similar tests described in the literature. The developed assay allows for the determination of CRP with a visual detection limit of 4 ng/mL in the required concentration range with acceptable intra-assay/inter-day reproducibility and selectivity.

Nanoparticles exhibiting peroxidase-like activity (nanozymes) are emerging as promising alternatives to enzyme labels in colorimetric assays, including ELISA. Unlike natural peroxidases, nanozymes demonstrate distinct mechanisms for oxidizing chromogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB), prompting the use of custom substrate formulations in contemporary research. However, analysis of the literature reveals that such compositions often yield abnormally high background signals due to non-specific oxidation of TMB, likely catalyzed by trace metal ions present in assay buffers. This phenomenon narrows the dynamic range, elevates limits of detection (LOD), and restricts the use of higher substrate concentrations that could otherwise enhance assay sensitivity. In this study, we identify the widespread use of sodium acetate buffer as a principal contributor to non-specific oxidation, owing to its limited chelating capacity. Statistical analysis demonstrates that lower pH, elevated TMB concentration, and, to a lesser extent, increased buffer molarity and hydrogen peroxide concentration are the most significant factors promoting non-specific reactions. Additional variables influencing the background include reagent purity and organic co-solvent content. Employing platinum nanozymes as a model system, we propose two effective strategies to mitigate non-specific oxidation: (i) substitution of acetate buffer with citrate buffer and (ii) supplementation of acetate buffer with chelating agents. These approaches reduce LOD by 3–12-fold while maintaining low background signals even under adverse conditions (high TMB concentration and low pH).

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.

Nanozymes, while promising alternatives to natural peroxidases in colorimetric assays, are often hindered by lower catalytic efficiencies. Although numerous approaches have been developed to improve signal intensity in nanozyme-based assays, optimization of the reaction medium in which the nanozyme interacts with the substrate remains a significantly underexplored area. The vast majority of studies rely on standard sodium acetate buffers or commercially sourced reagents optimized for horseradish peroxidase, neglecting the unique catalytic properties of different nanozymes. This work presents a systematic optimization of 3,3',5,5'-tetramethylbenzidine (TMB)-based reaction medium composition for four common nanozymes: iron oxide, LaNiO3, Mn-doped CeO2, and platinum nanoparticles. Our findings reveal that while sodium acetate buffer is suitable for LaNiO3, alternative buffers significantly enhance signal intensity (41-68%) for the other nanozymes. Further optimization of ionic strength, organic cosolvent type and concentration, and TMB/H2O2 concentrations yielded improvements in signal intensity, analytical sensitivity, and assay time. This study also identifies common pitfalls encountered during optimization of reaction conditions and proposes potential solutions. We posit that reaction medium should be a standard optimization step in the development of nanozyme-based assays, and the use of commercially sourced reagents with undisclosed compositions should be avoided.

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).

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. 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. We systematically optimized synthesis parameters 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.

Prussian blue nanoparticles (PBNPs), also called nanozymes, are very attractive as an alternative to horseradish peroxidase in immunoassay development due to their simple and low-cost synthesis, stability and high catalytic activity. Today, there is a method for highly effective PBNP synthesis based on the reduction of an FeCl3/K3[Fe(CN)6] mixture by hydrogen peroxide. However, there is a lack of research showcasing the use of these highly effective PBNPs for specific target detection in clinical settings, as well as a lack of comprehensive comparisons with conventional methods. To address this gap, we prepared diagnostic reagents based on highly effective PBNPs by modifying them using gelatin and attaching anti-C-reactive protein (CRP) monoclonal antibodies through cross-linking with glutaraldehyde. As a result, a solid-phase colorimetric immunoassay in a sandwich format (nanozyme-linked immunosorbent assay [NLISA]) using highly effective PBNPs as a label for CRP detection has been demonstrated for the first time. The assay demonstrated a detection limit of 21.8 pg/mL, along with acceptable selectivity, precision (CV < 25

LaNiO3 perovskite nanoparticles, especially nanospheres (LNNS), show great promise in biomedical assays due to their peroxidase-like catalytic properties. However, LNNS-based diagnostic reagents have not been tested in nanozyme enzyme-linked immunosorbent assay (NLISA) or other enzyme-linked immunosorbent assays, and there is limited data on their synthesis. To fill this gap, it is necessary to develop a method for creating LNNS conjugates with monoclonal antibodies and to investigate the reproducibility, scalability, and applicability of these diagnostic reagents in NLISA. We have successfully developed a method for producing novel diagnostic reagents utilizing LaNiO3 nanospheres. Our research demonstrates the application of these nanospheres in a NLISA specifically designed for the detection of C-reactive protein (CRP) in real serum samples. This method is both reproducible and scalable, allowing for the efficient production of nanospheres that are functionalized with monoclonal antibodies targeting CRP, with a mean diameter of approximately 270 nm. Based on the promising results obtained from our experiments, we have developed and optimized a sandwich-format NLISA for CRP detection. This assay achieved a lower limit of detection at 0.178 mu g L-1, with a dynamic range from 12.5 to 0.195 mu g L-1 and a linear detection range extending from 0.195 to 6.25 mu g L-1, showcasing its potential for clinical applications. The new NLISA method, utilizing LaNiO3 nanospheres in a sandwich format for the detection of CRP, significantly enhances sensitivity compared to similar use horseradish peroxidase-based ELISA. In this study for the first time, the functionalization of lanthanum nickelate nanospheres with recognition elements has been demonstrated. This advancement also sheds light on the technological challenges involved in synthesizing diagnostic reagents, identifying areas that need further exploration.

Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter Facebook Reddit LinkedIn Tools Icon Tools Reprints and Permissions Cite Icon Cite Search Site Citation S. Uzhviyuk, M. Bochkova, V. Timganova, P. Khramtsov, K. Shardina, V. Vlasova, E. Saidakova, S. Zamorina; PEGylated graphene oxide and monocyte metabolism. AIP Conf. Proc. 20 February 2024; 2924 (1): 050005. https://doi.org/10.1063/5.0182629 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAIP Publishing PortfolioAIP Conference Proceedings Search Advanced Search |Citation Search

Nanozymes, while promising alternatives to natural peroxidases in colorimetric assays, are often hindered by lower catalytic efficiencies. While various strategies exist to enhance signal intensity in nanozyme-based assays, substrate optimization remains largely underexplored. The vast majority of studies rely on standard sodium acetate buffers or commercially-sourced substrates optimized for horseradish peroxidase, neglecting the unique catalytic properties of different nanozymes. This work presents a systematic optimization of 3,3',5,5'-tetramethylbenzidine (TMB)-based substrate compositions for four common nanozymes: iron oxide, LaNiO3, Mn-doped CeO2, and platinum nanoparticles. Our findings reveal that while sodium acetate buffer is suitable for LaNiO3, alternative buffers significantly enhance signal intensity (41-68%) for the other nanozymes. Further optimization of ionic strength, organic co-solvent type and concentration, and TMB/H2O2 concentrations yielded improvements in signal intensity, analytical sensitivity, and assay time. This study also identifies common pitfalls encountered during substrate optimization and proposes potential solutions. We posit that substrate composition should be a standard optimization step in the development of nanozyme-based assays, and the use of commercially-sourced substrates with undisclosed compositions should be avoided.

Prussian Blue, a blue coordination polymer, emerges as a promising candidate in the realm of biomedicine. Its nanoparticles, known as catalytic labels or nanozymes, exhibit remarkable peroxidase-like properties and serve as effective antioxidants. Unsurprisingly, the demand for synthesizing Prussian Blue nanoparticles with customizable sizes is on the rise. In this study, we unveil a novel approach to synthesizing Prussian Blue nanoparticles. In this work, the synthesis of Prussian Blue nanoparticles by reducing an equimolar mixture of FeCl3 and K3[Fe(CN)6] with hydrogen peroxide in different water-alcohol mixtures was demonstrated for the first time. Alcohols with a lower dielectric constant (propanol-1, isopropyl alcohol, and tert-butanol) contribute to an increase in nanoparticle size, particularly at mole fractions of 0.02-0.05 and beyond. Conversely, alcohols with a higher dielectric constant (ethanol, methanol, ethylene glycol, and propylene glycol, excluding glycerol) demonstrate the ability to decrease nanoparticle size at mole fractions of 0.2-0.26 and higher. Building upon these findings, we present a scalable and reproducible method for preparing small Prussian Blue nanoparticles, measuring 30-40 nm, with enhanced peroxidase-like activity using 79.2% ethylene glycol as a solvent. The proposed mechanism behind the effect of ethylene glycol involves the limitation of both growth and secondary aggregation of Prussian Blue nanoparticles. These synthesized nanoparticles prove their efficiency as catalytic labels in a model vertical flow immunoassay designed to detect antibodies against SARS-CoV-2.

Background: LaNiO3 perovskite nanoparticles, especially nanospheres (LNNS), show great promise in biomedical assays due to their peroxidase-like catalytic properties. Methods for synthesizing LaNiO3 nanoparticles of various shapes have been developed, with spherical and rod-shaped LaNiO3 showing better stability. However, LNNS-based diagnostic reagents have not been tested in nanozyme enzyme-linked immunosorbent assay (NLISA) or other enzyme-linked immunosorbent assays, and there is limited data on their synthesis. To fill this gap, it is necessary to develop a method for creating LNNS conjugates with monoclonal antibodies and to investigate the reproducibility, scalability, and applicability of these diagnostic reagents in NLISA. Results: We have successfully developed a method for producing novel diagnostic reagents utilizing LaNiO3 nanospheres. Our research demonstrates the application of these nanospheres in a NLISA specifically designed for the detection of C-reactive protein (CRP) in real serum samples. This method is both reproducible and scalable, allowing for the efficient production of nanospheres that are functionalized with monoclonal antibodies targeting CRP, with mean diameter of approximately 270 nm. Based on the promising results obtained from our experiments, we have developed and optimized a sandwich-format NLISA for CRP detection. This assay achieved lower limit of detection at 0.178 µg/L, with a dynamic range from 12.5 to 0.195 µg/L and a linear detection range extending from 0.195 to 6.25 µg/L, showcasing its potential for clinical applications. Significance and Novelty: The new NLISA method, utilizing LaNiO3 nanospheres in a sandwich format for the detection of CRP, significantly enhances sensitivity compared to similar use horseradish peroxidase-based -ELISA. In this study for the first time first the functionalization of lanthanum nickelate nanospheres with recognition elements have demonstrated. This advancement also sheds light on the technological challenges involved in synthesizing diagnostic reagents, identifying areas that need further exploration.

We investigated the direct effect of PEGylated graphene oxide (P-GO) nanoparticles on the differentiation, viability, and cytokine profile of activated T helper type 17 (Th17) in vitro. The subject of the study were cultures of “naive” T-helpers (CD4+) isolated by immunomagnetic separation and polarized into the Th17 phenotype with a TCR activator and cytokines. It was found that P-GO at low concentrations (5 µg/mL) had no effect on the parameters studied. The presence of high concentrations of P-GO in T-helper cultures (25 μg/mL) did not affect the number and viability of these cells. However, the percentage of proliferating T-helpers in these cultures was reduced. GO nanoparticles modified with linear polyethylene glycol (PEG) significantly increased the percentage of Th17/22 cells in cultures of Th17-polarized T helpers and the production of IFN-γ, whereas those modified with branched PEG suppressed the synthesis of IL-17. Thus, a low concentration of PEGylated GO nanoparticles (5 μg/mL), in contrast to a concentration of 25 μg/mL, has no effect on the Th17-polarization of T helpers, allowing their further use for in-depth studies of the functions of T lymphocytes and other immune cells. Overall, we have studied for the first time the direct effect of P-GO nanoparticles on the conversion of T helper cells to the Th17 phenotype.

The interaction of graphene oxide nanoparticles with human peripheral blood mononuclear cells was studied using the Cell-IQ continuous monitoring system for living cells. We used graphene oxide nanoparticles of various sizes coated with linear or branched polyethylene glycol (PEG) in concentrations of 5 and 25 μg/ml. After 24-h incubation with graphene oxide nanoparticles, the increase in the number of peripheral blood mononuclear cells at visualization points decreased; nanoparticles coated with branched PEG more markedly suppressed cell growth in culture. In the presence of graphene oxide nanoparticles, peripheral blood mononuclear cells retained high viability in culture after daily monitoring in the Cell-IQ system. The studied nanoparticles were engulfed by monocytes and the type of PEGylation had no effect on this process. Thus, graphene oxide nanoparticles reduced the increase in peripheral blood mononuclear cell mass during dynamic observation in the Cell-IQ system without reducing their viability.

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.

Microalgae, in particular Chlorella vulgaris, are currently the most important tools for modern technological production of various products and goods. Over the past decades, the field of application of microalgae has significantly expanded and there is no doubt that microalgae-based technologies will develop and find new applications. The creation of renewable fuel feedstock from Chlorella vulgaris is estimated to help overcome the economic and technical problems associated with declining oil reserves. Much room for improvement of these technologies remains in the search for new ways to stimulate both physical (lighting, magnetic fields, temperature) and chemical (phytohormones, fertilizers, small organic molecules). This review paper will look into the prospects for industrial applications of Chlorella vulgaris, as well as ways to increase its biomass and beneficial metabolite content.

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.

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.

The aim of the study was to optimize the conditions for a model immunoassay in the immunofiltration format using diagnostic reagents based on horseradish peroxidase. Residual blood serum samples from patients in the red zone with a verified diagnosis of a new coronavirus infection were used as positive sera, and blood sera obtained before 2019 were used as negative samples. The procedure of immunofiltration analysis was carried out using a pool of negative and positive blood sera. Studies were carried out to optimize the analysis procedure and increase the significant characteristics of the test. Results. It has been shown that the addition of sodium dodecyl sulfate to a final concentration of 50 M in the substrate buffer makes it possible to achieve a higher analytical signal and a stable result 10 minutes after the end of the analysis procedure. Such conditions of immunofiltration analysis as dilutions of the diagnostic reagent, the volume of the introduced sample and the amount of the S-protein of the coronavirus applied to the nitrocellulose membrane were optimized. It has been determined that using immunofiltration analysis it is possible to detect antibodies against the coronavirus S-protein in a dilution of a serum sample of more than 1/1000. The results of immunofiltration analysis reproduce the results of ELISA.

The effect of graphene oxide (GO) nanoparticles of 100–200 nm in size coated with linear (LP-GO) and branched (BP-GO) polyethylene glycol at concentrations of 5 and 25 μg/mL on the metabolism of Jurkat tumor cells was studied. It was found that LP-GO nanoparticles at a concentration of 25 μg/mL can enhance basal glycolysis of Jurkat T-lymphocyte tumor cell line cells, while LP-GO and BP-GO at the same concentration can reduce the indicators of compensatory glycolysis. Despite this, GO nanoparticles coated with linear and branched PEG at a concentration of 5 μg/mL do not have pronounced effects on oxidative phosphorylation and glycolysis of Jurkat cells and could therefore be safe for activated T cells.