The use of a chemiluminescence in the assessment of the nanomaterials antioxidant activity

Abstract

Nanomaterials are one of the most promising classes of advanced materials with fine-tuned biological activities. This is evidenced by the presence of redox activity of a number of nanoparticles aimed at inhibiting free radicals and/or mimicking the functions of enzymes. At the same time, it is impossible to study the expression of these biological properties without the use of well-standardized, representative techniques that provide availability, high precision, sensitivity, and selectivity of the measured characteristics. A method that satisfies these requirements is chemiluminescence analysis, which is widely used both in clinical analysis and to characterize the antioxidant activity of substances of natural or synthetic origin. Recently, a trend of using chemiluminescence analysis to study the biological activity of nanomaterials has appeared as a suitable alternative to spectroscopic and electrochemical techniques. This review briefly describes the examples of successful applications of chemiluminescence methods to study radical-binding and enzyme-like activities of nanomaterials. We discuss the data about the effect of the used reagents (radical-generating systems, chemiluminescence activators) and experimental conditions on the obtained values characterizing the nanomaterials activity.

Graphical Abstract

Introduction

CL is the emission of light resulting from chemical reactions accompanied by the release of energy in the form of photons during the relaxation of the reaction product molecules from the electronically excited state to their ground state (Vladimirov and Proskurnina 2009). CL frequently accompanies processes in biological systems, especially in human and animal cells. Thus, the best known examples of CL in living organisms are the intense glow of bacteria of the family Vibrionaceae (genera Vibrio, Aliivibrio, Photobacterium) and some genera of the families Enterobacteriaceae and Shewanellaceae (Zavilgelsky and Shakulov 2018), as well as glow of dinoflagellates (Noctiluca scintillans, Pyrocystis lunula) (Valiadi and Iglesias-Rodriguez 2013), fungi (Bondar et al. 2012), and insects (Oleiwi et al. 2021). The intense luminescence emitted by these organisms is usually associated with the presence of specialized systems in their bodies, like “luciferin-luciferase” or “luciferin-luciferase-photoprotein”. Thus, the intense luminescence of fireflies is caused by the presence of the “luciferin-luciferase” system in their body. The redox transformations of this system, with the involvement of ATP and molecular oxygen, are associated with the emission of light quanta. Another example of CL in bio-objects is the low-intensity luminescence of tissues and cells caused by the generation of free radicals (O₂^∙-, O₂^*, ∙OH, etc.) in them. These radicals are generated in the processes of oxidation and auto-oxidation of various molecules, in the electron transport chain (terminal oxidation chain), during the functioning of various enzymes—oxidases, cyclooxygenases, lipooxygenases, and dehydrogenases (Fleiss and Sarkisyan 2019). Examples of this type of luminescence are CL during lipid peroxidation and activation of phagocytic cells (Vladimirov et al. 1992). A diversity of processes accompanied by CL in biobjects determines the prospects for the development of methods based on this phenomenon to diagnose their functional state or to study reactions characteristic of biological systems.

Use of chemiluminescence for biological molecule and nanomaterial analysis

Evaluation of the influence of the analyst on the CL intensity is the basis of a whole set of methods for quantitative and qualitative determination of chemical elements and compounds by CLA methods (Fig. 1). Advantages such as the absence of radioactive waste, relatively simple equipment requirements, very small detection limits, and a large dynamic range make CLA methods widely used in clinical analysis (Dodeigne et al. 2000). CLA is used in clinical practice for measurement of ATP, creatine kinase, adenylate cyclase, phosphodiesterase, ATP-ase, proteolytics, and other enzymes as well as their isoforms. The potential of CLA has especially expanded after the development of its modifications based on the immunoluminescent assay (Zhang et al. 2018). In addition, the CLA methods are one of the most standardized methods for determining the antioxidant properties of substances and simulate the ability of living cells to participate in photosensitizing processes. CL methods for determining ARA are based on the ability of radicals to luminesce in the recombination reaction as a result of optical excitation of photosensitizer particles, i.e., direct conversion of chemical energy into the energy of electromagnetic oscillations. CLA is widely used as a sensible method to observe the production or inhibition of free radicals. At the same time, the generation of free radicals is accompanied by the emission of light, which may be detected by luminometers. Figure 1 presents a summary of the main areas of CLA use. Due to its high sensitivity and expressivity, CLA can be used to assess the ARA and enzyme-like activity of a number of substances, including nanomaterials, which have been of increasing interest in recent years (Guo et al. 2007).

Fig. 1.

The main application fields for chemiluminescence analysis

It is known that reducing the size of particles to the nanometer range is accompanied by a number of fundamental changes in the properties of matter, in particular, by a change in the thermodynamic state of nanosystems compared to the classical bulk and by the phenomenon of quantum-dimensional effects (Jagiello et al. 2017). Due to their small size, they have a high value of the interface and an excess of surface energy, which results in the appearance of high physical and chemical activity of nano-objects. Interest in the involvement of nanoparticles in the development of drugs for biomedicine, veterinary medicine, and agriculture is due to their promising biological properties (antibacterial, antiviral, immunomodulatory, etc.), the expression of which depends on their nature, particle size, and nanomorphological characteristics (Sim and Wong 2021; Soares et al. 2018). During development of potential drugs for biomedical applications, special attention is paid to the means of combating free radicals, which are formed in excess in the process of oxidative stress. The result of this process is a disruption of the balance of concentrations of pro-oxidant and antioxidant components (enzymatic and non-enzymatic in nature) with the subsequent start of a cascade of pathological redox-dependent processes, the final result of which is damage to cell membranes (due to the development of lipid peroxidation processes) and the initiation of cell death mechanisms (necrosis, ferroptosis). The use of nanoparticles as potential antioxidants has received special attention in this respect (Vaiserman et al. 2020; Li et al. 2020). Today, in vitro, the pronounced radical-binding ability of a number of nanoparticles of different chemical nature—noble metals (Ag⁰, Au⁰, Pt⁰, Pd⁰), metal oxides (Fe₃O₄, ZnO, CeO₂, NiO, Cu₂O), and elemental chalcogenes (Se⁰), against free radicals 2,2-diphenyl-1-picrylhydrazyl, cation-radical 2,2'-azino-bis 3- ethylbenzothiazoline-6-sulfonate, NO^∙, and O₂^∙-, has been successfully demonstrated (Valgimigli et al. 2018; Lesnichaya et al. 2020; Kumar et al. 2020). Thus, the prospects for the use of cerium oxide nanoparticles in biomedicine are determined by their ability to bind ROS due to the facile and reversible change in their Ce⁺³ and Ce⁺⁴ oxidation degree depending on environmental conditions (Das et al. 2013). In the bulk state, cerium oxide can exist either as CeO₂ (Ce⁺⁴) or as Ce₂O₃ (Ce⁺³). The conversion of cerium oxide to the nanoscale state is accompanied by the appearance of both Ce⁺⁴ and Ce⁺³ on the surface of its nanoparticles. The increase in the relative number of atoms on the surface of the particle with a decrease in its size leads to a gradual decrease in the effective oxidation state of Ce as a result of the elimination of some oxygen atoms. A rather free transition between Ce⁺³ and Ce⁺⁴ is the reason for the instability of the oxygen stoichiometry of cerium oxide, which causes its high activity, especially in the presence of such reactive particles as radicals (Tsunekawa et al. 2000; Deshpande et al. 2005). According to the available data, cerium oxide nanoparticles are able to interact with a number of substances of radical nature or radical precursor molecules; in particular, Nelson et al. (2016) presented data about the ability of cerium oxide nanoparticles to neutralize O₂^·-, ^·OH, ^·NO, O₂NO^-, and H₂O₂. The direction of cerium redox transformation is determined both by the type of radical (reducing or oxidizing agent) and by such medium parameters as pH and the presence of H₂O₂ molecules or (PO₄)^3- ions in the medium due to their direct influence on the ratio of cerium forms on the Ce⁺³/Ce⁺⁴ nanoparticle surface. The antioxidant action of silver nanoparticles is presumably based on the release of ions from their surface into the solution (or intercellular medium), followed by neutralization of free radicals due to one-electron transfer (Bedlovicova et al. 2020) (Scheme 1).

Scheme 1.

Proposed red-ox processes during silver nanoparticles antiradical activity effect

Among the available methods for studying the radical-binding capacity of potential antioxidant substances, the CLA method should be singled out as a separate group (Romodin 2021, 2022). This method is advantageous in comparison to the photometric, chemical and electrochemical techniques commonly used. The main advantage of this method is its high sensitivity, specificity of modeling experimental conditions corresponding to natural biological ones, and the possibility to investigate in detail the rate of reactions involving free radicals and to obtain information on the activity of a potential antioxidant and its antioxidant capacity. This method is based on comparing the chemiluminescence of the radical-generating system in the absence and presence of antioxidants, the introduction of which induces a change in luminescence kinetics due to changes in the number of free radicals. The range of radical-generating systems that are used today is characterized by sufficient diversity and is represented as systems based on biological molecules proper (“horseradish peroxidase-H₂O₂”, “cytochrome C-cardiolipin-H₂O₂”, “hemin-hydrogen peroxide”, etc.), and systems based on low molecular weight organic substances, in particular 2,2-azo-bis-isobutyronitrile or ABAP which thermolysis leads to the formation of two free radicals (Naumova et al. 2021; Vladimirov and Proskurnina 2009). Due to the extremely low intensity of CL of almost all radical-generating systems, as well as the organisms’ own biochemiluminescence, as well as non-specificity of the registered CL depending on the radical type, it becomes obvious the need to use different enhancers (activators) of CL. The most commonly used CL activators today are luminol, isoluminol, lucigenin, fluorescein, coumarin, and anthracene derivatives (Alexeev et al. 2012; Romodin 2022). The addition of an activator to the radical-generating system can significantly increase the intensity of the detected CL. Moreover, this makes it possible to register the kinetics of CL from a specific type of free radical. All of the above-mentioned systems have proven to be good reagents for an available and express way to study the radical-binding action of potential antioxidants, including nanomaterials. To date, however, the use of the CLA method to investigate the antioxidant activity of nanoparticles has been limited to a few publications. This does not match the potential value of this method for a detailed study of the radical bonding capability of nanomaterials and is likely to be temporary.

Thus, in Sozarukova et al. (2020a, 2020b), the activated chemiluminescence method combined with mathematical modeling for the first time quantitatively evaluated the antioxidant properties and investigated the superoxide dismutase-like activity of citrate-stabilized cerium oxide nanoparticles. SOD activity was evaluated in a standard biochemical model of superoxide anion-radical generation based on the xanthine oxidase enzyme and the substrate xanthine, using SOD as a standard compound and lucigenin as a selective CL probe for superoxide anion radical. The ARA of CeO₂ nanoparticles was investigated in a luminol-activated system ABAP as a source of free radicals. The active radical scavenging action of CeO₂ nanoparticles against alkylperoxyl radicals generated as a result of ABAP thermolysis and interaction of the resulting free radicals with oxygen was detected. However, the registered value of the expression of this activity under the chosen experimental conditions for CeO₂ nanoparticles was 20-fold lower compared to the standard antioxidant Trolox, with a corresponding Trolox equivalent activity value for 1 μmol/l CeO₂ sol of 0.049 ± 0.004 μmol/l. Compared with SOD, the radical-binding activity of CeO₂ nanoparticles against superoxide anion radical was also 6 times lower. At the same time, the experimentally obtained values of the radical-binding capacity of CeO₂ nanoparticles did not correspond to the previously obtained values in other works, indicating pronounced antiradical properties of cerium dioxide nanoparticles that are several times greater than those of both Trolox and SOD (Valgimigli et al. 2018). The reason for such a significant discrepancy in the results obtained by the authors is assumed to be the effect on the kinetics of interaction between the surface of antioxidant cerium oxide nanoparticles with free radicals of macromolecules stabilizing the surface of thermodynamically unstable nanoparticles, and also the modeling of test conditions of antiradical activity as close to the natural as possible, namely, the use of buffer systems that have a direct effect on nanoparticle activity. Figure 2 shows the proposed mechanism of the ARA of cerium oxide nanoparticles against the radicals produced by ABAP thermolysis. In work of Mikheev et al. (2021), using the same “ABAP-luminol” system allowed to characterize the antioxidant potential (capacity and activity) of aqueous dispersions of fullerenes C₆₀ and C₇₀ and endofullerene Gd@C₈₂. The ARA was evaluated by TRAP, by the semisuppression concentration, and by the suppression area during the initial period. The authors show that the antioxidant capacity of the nanomaterials increased in the order Gd@C₈₂ < C₇₀ < C₆₀.

Fig. 2.

Proposed mechanism of the ARA of cerium oxide nanoparticles against the ABAP free radicals

In Zvereva et al. (2023), the authors characterized the antioxidant properties of 3–5 nm silver nanoparticles stabilized with arabinogalactan by the use of luminol as a chemiluminescence activator and radical-generating system “horseradish peroxidase - H₂O₂”. It was found that the addition of silver nanoparticles into the radical-generating system “horseradish peroxidase - H₂O₂” is accompanied by a pronounced decrease in the CL intensity. The fixed changes in the CL kinetics depended on both the percentage content of silver in the composite (0.5–3.5% Ag) and the concentration of aqueous solution of nanoparticles (0.1–5.0 mg/ml). It was found that the preliminary addition to the radical-generating system aliquot 0.1–0.4 mg/ml aqueous solutions of silver nanoparticles is characterized by the general saving type of the kinetic curve, namely, a sharp increase of CL intensity in the initial period of experiment with a smooth gradual decrease to background values within 25 min. However, the CL intensity was reduced compared to the control by 19–28% (Fig. 3a, b). A similar behavior, namely, the absence of a latent period (the period of initial inhibition of free radical formation due to the active involvement of the antioxidant in the reaction with them), and exclusively the reduction of chemiluminescence intensity are typical of weak antioxidants, such as ionol, diferol, and mexidol (Vladimirov et al. 2016). While an increase of the concentration of nanoparticles solutions up to 2.5–5 mg/ml was accompanied by a significant (up to 94–96%) decrease of CL intensity compared to the control in the initial period of time followed by a slight increase of the CL intensity to the level of 61% of control (Fig. 3c), almost complete quenching of CL in the initial period of time can likely be considered as a latent period in the action of potential antioxidant silver nanoparticles. So, in high concentrations of aqueous solutions, the action of nanoparticles is typical for medium strength antioxidants such as for example tocopherol. It should be noted that due to the different percentage of silver nanoparticles in the arabinogalactan matrix, its amount in the volume of the analyzed sample is also different. In particular, for composites containing 1.9 and 3.6% Ag, significant inhibition of chemiluminescence is observed from the first minutes of registration and is maintained throughout the observation period. Therefore, a direct effect of silver nanoparticles on the ARA of the obtained composites can be concluded.

Fig. 3.

Luminol-enhanced chemiluminescence curves of the radical-generating system “horseradish peroxidase-H₂O₂” in the presence of Ag⁰NPs-containing composites with varying amounts of silver in composition and in concentrations of aqueous solutions: 0.2 mg/ml (a), 0.4 mg/ml (b), and 3.3 mg/ml (c)

In addition, CLA methods are actively used to evaluate the ability of nanoparticles of some metal oxides and noble metals to mimic natural enzymes. At the same time, these materials, known as nanoenzymes or nanozymes, demonstrate relatively high stability and reproducibility of the characteristics in a wide temperature and pH range, as well as biocompatibility, possibility of additional surface functionalization, and low cost (Yu et al. 2020). Thus, in Sozarukova et al. (2020a, 2020b), the catalase/peroxidase-like activity of citrate-stabilized CeO₂ colloidal solution was studied using analytical model systems lucigenin/H₂O₂ and luminol/H₂O₂. In the luminol/H₂O₂ system, a dose-dependent increase of luminescence intensity was observed with increasing concentration of added citrate-stabilized CeO₂ nanoparticles. That indicates their exhibiting peroxidase activity. At the same time, in the absence of the catalyst, the reaction of luminol with H₂O₂ in alkaline medium was relatively slow and characterized by weak chemiluminescence intensity. The introduction of cerium dioxide nanoparticles into the radical-generating system catalyzes the interaction of hydrogen peroxide with luminol, enhancing its luminescence. It should be noted that the peroxidase-like effect of cerium dioxide nanoparticles appears in a fairly wide pH range—both in the usual range of values from 4 to 6 and at higher pH values (7.2–8.5), whereas the lucigenin/H₂O₂ system was characterized by stationary luminescence kinetics, which did not change when the CeO₂ nanoparticles solution was introduced into the system. Therefore, under the selected conditions, the authors established the absence of catalase properties of CeO₂ nanoparticles (Vinothkumar et al. 2019). The authors also found a determinant effect on the peroxidase-like activity of cerium oxide nanoparticles of the surface stabilizing ligand type. This is confirmed by a 6-fold decrease in peroxidase-like activity when the surface of cerium dioxide nanoparticles is stabilized by maltodextrin macromolecules or a two-fold increase when ammonium citrate is used as a stabilizing ligand. The detection of hydrogen peroxide (Li et al. 2018; Han et al. 2017), glucose (Lv et al. 2014), heavy metal ions (Kanan and Malkawi 2021), and immunoassay (Maji et al. 2015) has the greatest potential for practical use of the peroxidase-like activity of nanomaterials. Enzyme-like activity of cerium dioxide nanoparticles was also studied in the Co(II)/H₂O₂ system in the presence of the CL probe lucigenin. The chosen experimental conditions make it possible to simulate the presence of several types of free radicals simultaneously in the medium—superoxide anion radical and hydroxyl radical. It was found that an increase in the concentration of CeO₂ nanoparticles in the analyzed sample is accompanied by an increase of chemiluminescence intensity due to their peroxidase-like activity, i.e., in this system, cerium dioxide nanoparticles can act as pro-oxidants. At the same time, the pro-oxidant properties of nanomaterials provide prospects for their use as potential agents for the elimination of exogenous components (toxins, bacteria, and viruses) (Zhidkova et al. 2011)

In Sozarukova et al. (2021a, 2021b), the authors also investigated the enzyme-like activity of cerium dioxide nanoparticles; namely, they found lipoperoxidase and phospholipoperoxidase activities. At the same time, the expression of enzyme-like activity of cerium oxide nanoparticles was compared with the activity of compounds containing metal ions of variable valence (especially Fe⁺²)—hemoglobin and Fe(III)-carboxymaltose, because these ions play a key role in the formation of radicals and the development of lipid peroxidation. The authors found that the activity of CeO₂ nanoparticles significantly differed from the activity of the reference substances, despite the higher value of redox potential for Ce⁺⁴/Ce⁺³ pair (1.44 V) compared to the value of 0.77 V for Fe⁺³/Fe⁺² pair. The study was carried out using a system consisting of phosphate buffer solution with pH 7.4, coumarin as a CL probe sensitive to lipid or phospholipid radicals, and model lipid, linoleic acid hydroperoxide (LOOH), or phospholipid, phosphatidylcholine hydroperoxide (PCOOH), substrates. It was found that the addition of different concentrations of citrate-stabilized CeO2 nanoparticles to coumarin-LOOH or coumarin-PCOOH mixture is accompanied by a direct dose-dependent increase in CL intensity, which indicates the intensification of substrate decomposition with the formation of free radicals, i.e., the pronounced pro-oxidant effect of CeO2 nanoparticles. At the same time, unexpectedly low values of pro-oxidant activity of cerium oxide nanoparticles in comparison with iron compounds are attributed by the authors to the tropism of cerium oxide to phosphate groups present in the sample volume due to the analysis in phosphate buffer.

Thus, we briefly reviewed the main examples of using CL techniques to analyze the biological activity of nanomaterials. We demonstrated the prospects of these methods due to their accessibility, high sensitivity, simple data interpretation, and high selectivity, which allow to observe the rate of formation or inhibition of specific types of free radicals. A wide range of reagents makes it possible to vary the experimental conditions in a specific way and increase the sensitivity of the method specifically for each analyte. Nevertheless, in each specific case, before we assert the presence of antiradical activity in materials registered by chemiluminescent method, it is necessary to confirm the presence of this activity with the involvement of other methods, in particular spectrophotometric, voltammetric, and others. In addition, it is absolutely necessary to select optimal conditions for conducting experiments, in particular pH, solvent, type of buffer system, temperature, and other parameters that have a direct impact on the obtained results. Overall, the above-mentioned advantages of CLA allow us to hope for its widespread distribution for testing the biological activity of nanomaterials.

Abbreviations

CL

Chemiluminescence

CLA

Chemiluminescence analysis

ATP

Adenosine triphosphate

ROS

Reactive oxygen species

ARA

Antiradical activity

ABAP

2,2′-azo-bis 2-amidinpropane

SOD

Superoxide dismutase

TRAP

Total radical-trapping antioxidant potential

Author contributions

MVZ: writing-review and project administration. AVZ: visualization.

Funding

The study was supported by Russian Science Foundation (grant number 23-26-00140).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

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Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.