The Effect of Pristine and Pegylated Graphene Oxide Nanosheets on the Functions of Human Neutrophils

Graphene oxide (GO) is very useful for biomedicine, due to its physicochemical properties; therefore, its interaction with cells of the immune system has beenextensively studied. Many studies have aimed toreduce the undesirable effects of GO through chemical modification, including through polyethylene glycol (PEG) coating. Neutrophils are the first to respond to foreign object invasion in the body. Their main functions are the uptake and destruction of foreign particles, including with the help of reactive oxygen species (ROS).Our study aimed to investigate theengulfment of unmodified graphene oxide (GO) and graphene oxide coated with polyethylene glycol (GO-PEG) by human neutrophils and the effect of nanosheets on the production of ROS.We used sheets of GO (Ossila, Great Britain, average plate size 1-5 μm) and GO-PEG (569 ± 14 nm, PEG coating≈ 20%) at concentrations of 12.5μg/mL, 25μg/mL, and 50 μg/mL. The uptake of nanosheets was assessed by flow cytometry, taking into account the level of background adhesion of nanoparticles. ROS production was evaluated by luminol-dependent chemiluminescence (LCL).It was found that GO (12.5μg/mL, 25μg/mL, and 50 μg/mL) was actively internalized by neutrophils, while the uptake of GO-PEG was not detected. GO and GO-PEG particles (25 μg/mLand 50 μg/mL) reduced the total production of ROS by human leukocytes.Thus, the modifying of GOnanosheets with PEG resulted in the abolishment of their active uptake by neutrophils but did not affect the GO inhibitory effect on their oxidative activity. Keywords: graphene oxide surface modification, pegylated graphene oxide nanosheets, nanoparticle uptake, human neutrophils, of reactive oxygen specie

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 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 (CNP) 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 CNP 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 the 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 minutes) 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

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

Synthesis of Prussian Blue nanoparticles in water/alcohol mixtures

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

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

Albumin nanoparticles loaded with hemin as peroxidase mimics for immunoassay

Contemporary immunoassays commonly used in clinical diagnostics mostly utilize enzymes, such as horseradish peroxidase, for signal generation. Numerous research is dedicated to the development of artificial peroxidase-mimicking catalysts with lower cost, high activity, better operational stability, and tunable properties. Herein we synthesized hemin-loaded bovine serum albumin (BSA) nanoparticles and applied them as catalytic labels (nanozymes) in colorimetric immunoassay of anti-tetanus antibodies. Hemin is a key part of the peroxidase catalytic center, possessing peroxidase like-activity. Albumin nanoparticles were loaded with multiple hemin molecules and decorated with Streptococcal protein G. Resulting nanozymes possessed good colloidal stability and allowed for antibody detection in blood serum. The sensitivity of antibody detection was sufficient for the assessment of post-vaccination immunity

Synthesis and Application of Albumin Nanoparticles Loaded with Prussian Blue Nanozymes

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

Measuring the Concentration of Protein Nanoparticles Synthesized by Desolvation Method: Comparison of Bradford Assay, BCA Assay, hydrolysis/UV Spectroscopy and Gravimetric Analysis

Research paper on sunthesis of protein nanoparticlesAbstractThe 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

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 synthesis of ‘artificial peroxidase’ nanozymes. 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 allowing the obtaining of nanozymes with enhanced catalytic activity. ‘Artificial peroxidase’ 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

The Effect of PEGylated Graphene Oxide Nanoparticles on the Th17-Polarization of Activated T Helpers

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