Electrochemical micro- and nanobiosensors for in vivo reactive oxygen/nitrogen species measurement in the brain

Fatemeh Beigloo; Samira Amiri Khoshkar-Vandi; Elham Pourmand; Mona Heydari; Fatemeh Molaabasi; Nima Gharib; Yasser Zare; Kyong Yop Rhee; Soo-Jin Park

doi:10.1515/ntrev-2023-0124

Article Open Access

Electrochemical micro- and nanobiosensors for in vivo reactive oxygen/nitrogen species measurement in the brain

Fatemeh Beigloo , Samira Amiri Khoshkar-Vandi , Elham Pourmand , Mona Heydari , Fatemeh Molaabasi , Nima Gharib , Yasser Zare , Kyong Yop Rhee and Soo-Jin Park

Published/Copyright: October 25, 2023

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information

From the journal Nanotechnology Reviews Volume 12 Issue 1

Abstract

In this focused review, we examine the influence of reactive oxygen and nitrogen species (ROS/RNS) on physiological processes and the induction of oxidative stress, with particular emphasis on the brain and neuronal systems. We discuss the formation mechanisms of ROS and RNS, their significance in the brain, and various detection methods. The review investigates the latest advancements in nano-engineered electrochemical biosensors designed for in vivo monitoring of ROS and RNS in the brain tissue. We explore the electrochemical measurement of specific species, such as H₂O₂, superoxide, NO, and peroxynitrite, while providing a comparative evaluation of sensor designs for ROS and RNS detection in the brain. Finally, we offer an outlook and conclusion on the future of this field.

Keywords: electrochemical; biosensors; oxygen species

1 Introduction

Reactive oxygen and nitrogen species (ROS and RNS) are critical modulators of physiological and pathological processes in the brain. These species are generated through redox reactions involving molecules containing oxygen or nitrogen [1,2]. ROS, which encompasses superoxide (O₂ ^•), peroxyl radical (ROO^•), hydroxyl (HO^•), hydrogen peroxide (H₂O₂), hypochlorous acid/hypochlorite (HOCl/⁻OCl), and singlet oxygen (¹O₂) [1,2], and RNS, which include nitric oxide (^•NO), nitrogen dioxide (^•NO₂), and peroxynitrite (ONOO⁻), are consistently generated during regular cellular functions like oxidative phosphorylation, fatty acid catabolism, phagocytosis, macromolecular compound degradation, and protein folding processes [3,4].

At low or moderate levels, ROS and their derivatives serve vital physiological functions, including migration, differentiation, proliferation, hypertrophy, cytoskeletal dynamics, and metabolism [5,6,7]. For example, hydroxyl radicals (^•OH) as superoxide anion (O₂ ^−•) derivatives stimulate guanylate cyclase and activate the second messenger cyclic guanosine monophosphate formation, while hydrogen peroxide triggers the transcription factor nuclear factor kB (NF-kB) in various mammalian cells [8,9,10]. Moreover, during the inflammatory response, neutrophils and macrophages produce superoxide anions and other ROS/RNS to eliminate engulfed bacteria during the oxidative burst [11,12,13]. In the brain tissue, microglia and astrocytes generate ROS/RNS to modulate synaptic and non-synaptic communication between glia and neurons [14,15,16]. Additionally, RS interferes with increased neuronal activity and can induce synaptic plasticity, memory consolidation, and learning, which are altered by aging [17,18,19,20]. Furthermore, ROS/RNS regulates vascular tone through smooth muscle relaxation under normal physiological conditions [4,21]. However, excessive RS levels can cause oxidative damage to DNA, lipids, and proteins, promoting various diseases in affected tissue [22,23,24,25]. Thereby their levels must be tightly regulated to maintain cellular homeostasis.

Despite extensive research, the dual nature of ROS/RNS and their physiological and pathological processes in the brain and nervous system remain incompletely understood. This knowledge gap is primarily due to the challenges associated with in vivo detection of ROS/RNS species, given their extreme reactivity and short lifetimes [26,27]. Consequently, developing reliable and effective in vivo analytical methods to quantify ROS/RNS species and elucidate their pathological and physiological roles is of paramount importance. In this regard, the present study focuses on the application of electrochemical biosensors for in vivo quantification of ROS/RNS in the brain that consumes 20% of the body’s oxygen (O₂), the main source of ROS, due to its high rate of metabolism. We will discuss the mechanism of ROS/RNS formation and then the importance of these reactive species in brain functions and their in vivo measurement. Then, we discuss the current recent approaches to utilizing electrochemical biosensors for in vivo assessments of ROS/RNS in the brain. Eventually, a brief comparison between the electrochemical technique and other analytical techniques for ROS/RNS measurement will be discussed.

2 Formation mechanisms of ROS and RNS

The generation of ROS stems from endogenous and exogenous sources: Endogenous sources include biological processes that release ROS as by-products, such as the mitochondrial electron transport chain, the endoplasmic reticulum (ER), nitric oxide synthases (NOSs), membrane-bound NADPH oxidase (NOX) family enzymes, and microsomes and peroxisomes. Exogenous sources consist of cellular responses to bacterial invasions, cytokines, and xenobiotics due to oxidative burst activity in macrophages [28,29,30,31].

A considerable percentage of cellular free radicals are generated through mitochondrial oxidative phosphorylation as a result of electron leakage from the electron transport chain [32,33]. Mitochondria mainly produce metabolic energy as adenosine triphosphate (ATP) via oxidative phosphorylation following the oxidation of reduced co-enzymes involved in the respiratory chain. In this context, the superoxide radical is initially formed at complexes I and III of the electron transport chain by single electron reduction of O₂ through the H⁺ pumps of the respiratory chain on redox-active prosthetic groups of electron-binding proteins like reduced coenzyme [28,30,34]. This reduction is more favorable thermodynamically, enabling numerous electron donors within the mitochondria to support the reaction [34]. Approximately 1% of total mitochondrial O₂ consumption is utilized for generating the superoxide radical [5,33]. Electron leakage from redox centers or enzyme subunits or complexes leads to ROS formation in the mitochondrial matrix or intermembrane space. The superoxide radical is rapidly converted by the superoxide dismutase (SOD) enzyme into the non-radical ROS, H₂O₂, which passes through the membrane and disperses among cellular compartments [28,31,32,34]. Following this, the hydroxyl radical is formed through a respiratory burst via the Fenton reaction or the Haber–Weiss reaction from H₂O₂ and metal species such as copper or iron [33,35,36].

Moreover, the myeloperoxidase–halide–H₂O₂ system can also generate ROS. In the presence of the chloride ion, H₂O₂ transforms into hypochlorous acid. Enzymes like α-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes play a role in ROS production within mitochondria, while the monoamine oxidase enzyme family stimulates hydrogen peroxide formation through monoamine catabolism in neuromodulator and neurotransmitter homeostasis [32]. Other significant endogenous sources of ROS consist of NOX, xanthine oxidase, cytochrome P450, and lipoxygenases [28]. NOX family enzymes facilitate the creation of superoxide anions from O₂, subsequently generating ROS [33,37]. When activated, NOX1-3 and NOX5 mainly generate superoxide, while NOX4, DUOX1, and DUOX2 produce H₂O₂ directly [28,37]. Factors involved in ROS production through NOX family activation include environmental factors and various cytokines and hormones (Figure 1) [31,37]. Several research has demonstrated increased NOX levels following brain injuries such as traumatic brain injury (TBI), stroke, epilepsy, and hypoglycemia [38,39,40,41]. Notably, inhibition of NOX through pharmacological and genetic means has been shown to significantly mitigate secondary neuronal damage, highlighting the crucial role of NOX in the initiation and progression of pathological conditions [42,43]. Importantly, there exists a close correlation between ROS levels and NOX activity, with the degree of ROS increase being closely associated with patient prognosis [44]. Consequently, therapeutic strategies aiming to improve patient outcomes have explored the administration of antioxidants to neutralize ROS [45].

Figure 1

Some of the major endogenous pathways of ROS and RNS production.

Mitochondria also play a role in generating RNS (Figure 1). Nitric oxide, a crucial RNS, participates in many essential functions and is produced in the mitochondria through the conversion of l-arginine to l-citrulline, consuming molecular oxygen and NADPH during the process [17,29,30,33,36]. NOS isoforms catalyze this reaction, with their activity heavily reliant on the availability of NADPH, tetrahydrobiopterin, and molecular oxygen [17,46]. Three primary isoforms of NOS exist neuronal NOS (nNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2) [47]. The nNOS and eNOS are constitutively expressed in neurons and endothelial cells, respectively, while iNOS expression is triggered by inflammatory stimuli in various cell types, such as macrophages, smooth muscle cells, and astrocytes [48]. Under normal conditions, nitric oxide serves as a vasodilator, neurotransmitter, and immune response mediator. However, when concentrations increase, nitric oxide reacts with the superoxide anion to produce peroxynitrite, another highly reactive RNS, and powerful oxidant that can induce cell damage and inflammation [4,49]. Peroxynitrites can interact with lipids, DNA, and proteins, resulting in cellular dysfunction and tissue damage [50,51].

Besides mitochondria, peroxisomes and ER are other important intracellular organelles involved in ROS/RNS production. Peroxisomes are involved in ROS production through β-oxidation of very long-chain fatty acids, catabolism of purine and polyamine, as well as the metabolism of d-amino acids and polyunsaturated fatty acids [52]. It is worth mentioning that while the β-oxidation of very long-chain fatty acids is not considered a typical mechanism in the brain tissue [53], recent studies have revealed that astrocytes possess some capacity for β-oxidation of fatty acids [54,55,56]. This capacity, along with the involvement of fatty acid oxidation in the regulation of adult neuronal stem cell activity and axonal mitochondrial trafficking, suggests that the metabolism of fatty acids in the brain extends beyond neuronal energy metabolism [57,58]. Therefore, while β-oxidation of very long-chain fatty acids may not be a predominant pathway in the brain tissue, it is important to consider the potential contribution of astrocytic metabolism and its implications for ROS production. Furthermore, peroxisomal, such as xanthine oxidase and acyl-CoA oxidase, generates superoxide and H₂O₂ [59,60], implicating the capability of peroxisomes to facilitate the dynamic rotation of ROS generation and removal [61].

In the ER, protein folding and maturation processes involve the formation of disulfide bonds, which can lead to the generation of ROS. The ER oxidoreductin 1 enzyme transfers electrons from protein disulfide isomerase (PDI) to molecular oxygen, producing hydrogen peroxide in the process. There are several associations between ER stress and oxidative stress, and brain physiological conditions. For instance, the increased S-nitrosylation of PDI was observed in brains of people with Alzheimer’s disease (AD) compared to the healthy individual [62]. This redox modification occurring under ER stress conditions may contribute to increased ROS production and potentially contribute to the aggregation of amyloid beta (Aβ), a hallmark feature of AD [63]. Moreover, the aggregation of Aβ in AD exacerbates ER stress, leading to alterations in the morphology of both the ER and mitochondria [64]. These morphological changes further impact mitochondrial function, resulting in the dissipation of the mitochondrial membrane potential and the accumulation of mitochondrial ROS [63,64]. To counteract the detrimental effects of ER stress-associated ROS, several agents have shown promise in reducing or blocking ROS production. Glutamine, as well as antioxidants such as α-tocopherol, ascorbic acid, and β-carotene, has demonstrated protective effects by reducing ROS levels [65,66,67]. These agents play a crucial role in preserving neuronal cell integrity and defending against the damaging effects of free radicals. In addition to the complex interplay between ER stress, ROS production, and brain physiological processes, it is worth noting that NOS enzymes, present within the ER, have been identified in both neurons and glial cells in the nervous system [68,69]. These enzymes contribute to the generation of RNS, particularly nitric oxide, which can further impact cellular signaling and oxidative stress pathways. The involvement of NOS-mediated RNS production within the ER adds another layer of complexity to the intricate network of molecular events occurring in various brain conditions.

3 The importance of ROS and RNS in the brain

The brain has high susceptibility to oxidative damage stems from a combination of physiological, anatomical, and functional factors. Although the brain constitutes only 2% of the human body weight, it consumes 20% of total basal oxygen (O₂) due to its high metabolic rate [70]. This disproportionate O₂ consumption results in higher availability of O₂, the primary precursor of ROS. The majority of O₂ is consumed to generate ATP, which is required for energy-intensive processes such as action potentials, synaptic machinery, neurotransmission, and enzymatic reactions.

Although the brain possesses a complex neuroarchitecture consisting of diverse cell types and functional circuitry, the reasons behind its high energy demands remain elusive [70]. The brain’s reductive environment, marked by a comparatively low partial pressure of O₂, is likely intended to minimize ROS production. However, endogenous factors, including weak antioxidant defenses, restricted regenerative abilities, glymphatic waste removal, dependence on excitotoxic and auto-oxidizable neurotransmitters, the vulnerability of polyunsaturated fatty acids to peroxidation, calcium load, and the presence of redox-active metals, render the brain highly susceptible to oxidative harm [5,6,7]. Moreover, research has shown that ROS and RNS play a role in various brain injury mechanisms, such as mitochondrial dysfunction, proteasomal dysfunction, and inflammation [3,4]

The uncontrolled level of RS in the brain results in several diseases including, but not limited to, acute injury of the brain (brain cerebral and trauma ischemia), psychiatric disorders (schizophrenia, autism, depression, and attention deficit hyperactivity disorder), and chronic diseases (AD and Parkinson’s disease [PD]) (Figure 2) [14,71,72]. For instance, postmortem analysis of the brains of PD patients has unveiled a significant increase in markers indicative of lipid peroxidation, namely malondialdehyde and 4-hydroxynonenal. Moreover, evidence of oxidative damage to proteins, presented as protein carbonyls, is also observed [73]. Furthermore, research reports a notable rise in mitochondrial DNA common deletions within the remaining dopaminergic neurons of the substantia nigra in PD patients. These deletions are often attributed to the adverse effects of oxidative stress [74]. Studies using both toxin and genetic models of PD have illustrated an enhanced oxidative stress scenario, with ties to mitochondrial function. Mitochondrial complex I inhibitors, such as rotenone or 1-methyl-4-phenylpyridinium (MPP+), that induce neurotoxicity in dopaminergic neurons, have found to primarily provoke oxidative stress rather than causing metabolic alteration, as their harmful effects can be effectively mitigated by antioxidant treatment [75]. Increased level of myeloperoxidase, generated by reactive astrocytes, has also been observed in PD. This increase could lead to a surge in reactive hydroxyl (OH) and other radicals, which in turn can potentially drive neuronal loss [76]. Additionally, the upregulation of NADPH-oxidase, an enzyme implicated in both inflammation and ROS generation, has been reported in PD. This upsurge is associated with microglial activation, localized increase in ROS, and subsequent degeneration of dopaminergic neurons [77]. Mutations in the Parkinson protein-1 (DJ-1) gene have also been linked with a rare form of autosomal-recessive PD. The resultant loss of DJ-1 function triggers oxidative stress, thereby reinforcing DJ-1 neuroprotective role through its antioxidant mechanism within mitochondria [77,78]. In DJ-1 knockout mice, a notable increase in mitochondrial oxidant stress is observed, along with downregulation of mitochondrial uncoupling proteins [79].

Figure 2

Schematic representation of oxidative stress that could cause different neuronal-related diseases.

The ROS involvement in AD, similar to PD, has been under extensive investigation [80,81,82]. Several studies have emphasized the contribution of superoxide anion, hydroxyl radical, hydrogen peroxide, and nitric oxide to the oxidative stress-mediated neurodegeneration characteristic of AD [83,84]. It is also found that oxidative stress, a key driver of ROS, is observable even before the onset of symptomatic AD. Notably, this oxidative damage is not confined to the brain regions commonly vulnerable to AD but extends to peripheral regions as well [85,86,87]. Aβ, a key player in AD pathology, is reported to generate hydrogen peroxide (H₂O₂) from oxygen (O₂) through electron transfer processes involving redox-active copper(ii) and iron(iii) ions [88,89]. The produced H₂O₂ can then be readily transformed into a more reactive hydroxyl radical via Fenton chemistry. The levels of H₂O₂ produced by Aβ can be further amplified by co-incubating the peptide with a reducing substrate, such as cholesterol [88,90]. This finding demonstrates how ROS production can be modulated within the context of AD.

Additionally, oxidative stress is strongly associated with tau pathology, another hallmark of AD. Cells overexpressing tau protein exhibit increased vulnerability to oxidative stress, possibly due to peroxisome depletion [91,92]. Moreover, tau protein has been shown to induce ROS production in mitochondria. A clear instance of this is seen in tau transgenic mice carrying the P301L mutation, where hippocampal tau phosphorylation incites mitochondrial dysfunction, leading to H₂O₂ production, lipid peroxidation, and ultimately neuronal loss [91,92,93]. Intriguingly, a decrease in cytoplasmic superoxide dismutase 1 (SOD1) or a deficiency in mitochondrial SOD2 escalates tau phosphorylation in Tg2576 AD transgenic mice [94]. Moreover, reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase and mitochondrial dysfunction have been observed in the tau transgenic AD mouse model. This model, especially in aged animals, presents a scenario of increased ROS production, compromised ATP synthesis, and diminished mitochondrial respiration [95]. These findings emphasize the importance of understanding oxidative stress in different brain conditions for the development of potential therapeutic strategies.

One example of these therapeutic approaches is using antioxidants. It is crucial to emphasize that the primary objective of antioxidant therapy in disease states is to normalize increased ROS levels and minimize oxidative damage rather than interfering with ROS’s normal physiological roles. In this regard, animal models with deficiencies or overexpression of cellular antioxidant enzymes can offer strong support for targeting oxidative stress in neurological disorders. For instance, overexpressing SOD or catalase enzymes in animals has shown neuroprotection against various brain disorders and cognitive dysfunctions [96,97,98,99,100]. Similarly, studies highlighting deficiencies of compartmentalized antioxidants, including Sod1, Sod2, and Sod3, have proven valuable [96,97,99,101]. However, before considering antioxidant therapy, it is essential to determine if ROS and oxidative damage are merely correlated with the disease process or if they play a causative role. To address this question, it is necessary to consider several important criteria for assigning a causative role to ROS/RNS and antioxidant properties. These include identifying the specific RS associated with the disease process and assessing whether there is a strong rationale for ROS/RNS involvement, among other factors. By understanding these aspects, we can further emphasize the importance of better monitoring ROS and RNS in brain disorders and their potential therapeutic implications.

4 ROS/RNS detection methods

The process of detecting and quantifying ROS and RNS has been marked with difficulties, which arise from their inherent properties such as short lifespans, low physiological concentrations, and high reactivity. A range of analytical techniques, encompassing gas or liquid chromatography, fluorescence spectrometry, UV/Vis spectroscopy, electron paramagnetic resonance (EPR), and colorimetric sensors, have been utilized to identify and quantify ROS/RNS species, although these are beyond the scope of this review [102,103,104,105,106,107,108]. However, many of these methods fall short in providing concentration profiles for ROS/RNS species at their point of production, as they mainly rely on indirect quantification routes that require extended assay times. The principles, strengths, and weaknesses of each technique are presented in Table 1. Fluorescence imaging probes, for instance, frequently encounter issues like irreversible activation, and complex quantitative analysis due to problems in calibrating fluorescent dye intensity [26,109,110]. Thus, while fluorescent imaging has been used to visualize relative changes in H₂O₂ in the biological environment; the absolute concentrations of H₂O₂ cannot be determined using this method [111,112]. Also, while the EPR technique offers greater selectivity by differentiating molecules containing unpaired electrons through analyzing the electron paramagnetic spectrum, it necessitates stringent experimental conditions, such as a vacuum environment and the elimination of any internal magnetism, limiting its in vivo application [106].

Table 1

Comparison of different types of sensors for ROS/RNS measurement

Method	Principle of method	Strength	Weakness	Ref.
UV-Vis spectrometry	Absorbance of light by molecules at specific wavelengths	Simple, fast, and inexpensive	Limited selectivity and sensitivity, not suitable for in vivo detection, and low spatial/temporal resolution	[104]
Chromatography	Separation of analytes based on their interaction with stationary and mobile phases	High selectivity and versatility	Requires extensive sample preparation, expensive instrumentation, limited in vivo detection, moderate spatial/temporal resolution	[108]
Fluorescence	Emission of light after excitation at specific wavelengths	High sensitivity and specificity, suitable for in vivo detection, and high spatial/temporal resolution	Potential interference from other fluorophores, photobleaching	[102,103,113]
Chemiluminescence	Emission of light during chemical reactions	High sensitivity, no need for external light source, possible to do in vivo detection	Limited selectivity, prone to interference, moderate spatial/temporal resolution	[114]
EPR	Detection of unpaired electrons in molecules	Direct measurement of free radicals, high specificity	Requires specialized equipment, complex data analysis, limited in vivo detection, and moderate spatial/temporal resolution	[106]
Colorimetric sensor	Colorimetric reactions form a colored product with a chromogenic reagent	Low-cost, timesaving, simple, naked-eye observable, rapid	Relatively low sensitivity, strong background color, not suitable for in vivo detection, and low spatial/temporal resolution	[107]
Electrochemistry	Measurement of redox reactions at electrode interfaces	Real-time measurement, high sensitivity, in vivo detection, and high spatial/temporal resolution	Limited selectivity, electrode fouling, invasive	[103]

The electrochemical-based sensors, on the other hand, show promise for real-time monitoring of various species with exceptional sensitivity [115,116,117,118,119,120,121]. Moreover, the possibility of decreasing the electrode size to micro and nano levels provides several advantages, including (i) heightened sensitivity and ultra-low detection limits, (ii) superior selectivity, (iii) compatibility with in vivo measurements owing to their minuscule size, and (iv) cost-effectiveness [3,122,123,124]. The utilization of these devices facilitates investigations into the ROS/RNS mechanism of action and may resolve the ongoing debate surrounding ROS/RNS pathological and physiological roles. As a result, this concise review highlights recent developments in the use of electrochemical biosensors, either constructed with nanosized electrodes or modified with nanomaterials, to study oxidative stress and inflammation in the brain via in vivo quantification of essential ROS/RNS.

5 Electrochemical measurement of ROS in the brain

H₂O₂ and superoxide (O₂ ^∙) can be considered as the most important ROS that have been found to play a vital protective role as an intracellular messenger at normal physiological levels [125,126,127,128]. For instance, H₂O₂ can modulate neuroplasticity and synaptic transmission in the rodent brain [17,129]. Also, endogenous H₂O₂ modulates dopamine (DA) release through the activation of potassium ion-sensitive ATP channels in neurons’ presence at the substantia nigra and striatal [130,131]. Thus, in this section, recent studies on the in vivo electrochemical measurement of H₂O₂ and O₂ ^∙ as indicators of oxidative stress in the brain are investigated. It should be noted that most studies are focused on H₂O₂ measurement rather than O₂ ^∙. This is because (i) O₂ ^∙ is converted to hydrogen peroxide by SOD enzyme; (ii) H₂O₂ can accumulate to relatively higher concentrations, and (iii) H₂O₂ is more stable than O_2∙ and easier to determine [132]. It is also worth mentioning that the superoxide could also convert to peroxynitrite, and thus the H₂O₂ level might not solely represent the actual level of superoxide (Figure 1).

5.1 Electrochemical measurement of H₂O₂ in the brain

Recently, an electrochemical biosensor for in vivo monitoring of H₂O₂ in the brain has been introduced to fabricate this biosensor, first, Prussian blue (PB) was electrodeposited onto carbon nanotube (CNT) assembled carbon fiber microelectrodes (CFMEs), and then a thin layer of polydopamine (PDA) was coated on the as-prepared CFMEs through self-polymerization (Figure 3a and b). The PDA membrane improved the PB-based electrode stability during the electrocatalytic reduction of hydrogen peroxide in both in vitro and in vivo. The as-fabricated biosensor shows excellent selectivity, for in vivo quantification of H₂O₂, and is found to be interference-free from O₂ and many other electroactive species which coexist in the brain (Figure 3c). The amperometric response obtained at the as-fabricated electrode in the cortex of anesthetized rats, during local microinfusion of mercaptosuccinate (MCS) (Figure 3d) and electrical stimulation (Figure 3e), confirms the ability of microelectrode for in vivo monitoring of hydrogen peroxide level during pathological and processes associated with drug infusion and electrical stimulation [133].

Figure 3

In vivo ROS biosensors: (a) Schematic representation of microbiosensor fabricated by the PDA/PB/CNT/carbon fiber electrode and implanted in rat brain. (b) The scanning electron microscopy (SEM) image of microelectrode modified by the PB/CNT/carbon fiber electrode. (c) Cyclic voltammetry gained at the PDA/PB/CNT/carbon fiber electrode in rat cortex. (d) The amperometric response obtained at the PDA/PB/CNT/carbon fiber electrode in the cortex of anesthetized rats where two different concentrations of mercapto succinate, 1 and 10 nM, are locally microinfused in the cortex. (e) The amperometric response measured at the PDA/PB/CNT/carbon fiber in the cortex of anesthetized rats when electrical stimulation of 5 V was applied for 10 s. Note that the electrode was polarized at −0.05 V vs Ag/AgCl [133].

While the proposed design by Li et al. holds promise for tracking changes in H₂O₂ levels in vivo during various physiological and pathological processes, there may be some potential drawbacks or limitations to consider. For instance, although the study demonstrates good stability for the sensor in vitro and in vivo, long-term stability and performance were not addressed. The biosensor’s effectiveness over extended periods of time needs to be evaluated. Furthermore, the sensitivity of the biosensor might be affected by changes in the brain environment during various physiological or pathological processes, which could potentially impact the accuracy of the H₂O₂ measurements. In addition, the fabrication process of the biosensor, which involves electrodeposition and self-polymerization, might face challenges in terms of scalability and reproducibility. Also, the PDA membrane has good biocompatibility, more in-depth assessments of its long-term biocompatibility and potential immune response are necessary for broader applications in vivo.

Simultaneous monitoring of neurotransmitters, such as DA, and ROS, like hydrogen peroxide (H₂O₂), is crucial for understanding the complex interactions and dynamics within the brain [134]. Both neurotransmitters and ROS play essential roles in normal brain function and have been implicated in various neurophysiological and neuropathological processes. By tracking both neurotransmitters and ROS, researchers can gain insights into the complex interactions and dynamic changes in the brain, which may lead to the development of novel therapeutic strategies for neurodegenerative diseases and other neurological conditions. In this regard, Zhang et al. developed a novel ring-disk microelectrode for the simultaneous in vivo electrochemical detection of H₂O₂ and DA [134]. The carbon fiber disk electrode was modified with PB and poly(2,3-dihydrothieno-1,4-dioxin) (PEDOT) to selectively detect H₂O₂, while the gold (Au) ring electrode was used for DA detection. The microelectrode exhibited a sensitive response to both H₂O₂ and DA with detection limits of 0.4 and 0.18 μM, respectively. The developed ring-disk microelectrodes were successfully employed for in vivo measurements of H₂O₂ and DA in the rat brain, providing a new platform for studying the neurophysiology and pathology of these neurochemicals. This approach demonstrates significant advancements in simultaneous in vivo detection of H₂O₂ and DA; however, some limitations and potential improvements should be considered. The study did not observe simultaneous changes in H₂O₂ and DA during the electrical stimulation process. Future research should investigate other physiological or pathological processes where such simultaneous changes might occur. Additionally, the selectivity and sensitivity of the microelectrode could be further enhanced by exploring novel materials or electrode configurations to improve the detection of other neurochemicals or multiple substances simultaneously.

Another important consideration that helps in better electrochemically quantifying the ROS level is considering the response change due to the pH [135]. The pH level could not only affect the electrochemical signal but also could give some hints on the physiological condition of the brain. Thus, simultaneously measuring ROS and pH in the brain is also important for understanding various neurological disorders and their progression. ROS and pH are interdependent factors that play crucial roles in neuronal function and dysfunction. ROS overproduction and pH imbalance are associated with several neurodegenerative diseases, such as ischemia. Tian group developed a selective and accurate ratiometric electrochemical biosensor for detecting H₂O₂ and pH through both current and potential outputs in a rat brain following ischemia [135]. Catalase (Cat) was used as a specific recognition element for both H₂O₂ and pH, while ferrocene (Fc), an insensitive molecule for H₂O₂ and pH, was used as a built-in correction to improve accuracy. The electrode exhibited high sensitivity, linearity, and selectivity for the analysis of H₂O₂ and pH, making it useful for monitoring their changes in rat brains following cerebral ischemia. The developed biosensor was modified with single-walled CNTs (SWNTs) assembled on a CFME, which improved the direct electron transfer of Cat for detecting H₂O₂ and pH. The selectivity of the Cat/Fc/SWNT/CFME electrode was evaluated, and negligible change in the signal was observed after metal ions, amino acids, and neurotransmitter species with biological concentrations were added. The advanced ratiometric H₂O₂ and pH biosensor, featuring exceptional selectivity and precision, together with the innate characteristics of CFMEs, such as miniaturization and excellent biocompatibility, offered a dependable platform for in vivo measurement of H₂O₂ and pH in the rat brain post-ischemia. Baseline levels of pH and H₂O₂ were determined in the striatum, hippocampus, and cortex of a healthy rat brain, and the alterations in these areas following cerebral ischemia were effectively tracked [135]. However, there are also some potential drawbacks to consider. Besides the invasive implantation, the sensor accuracy may be affected by the surrounding microenvironment and the stability of the sensor over time. Finally, the high cost and complexity of the system may limit its accessibility to researchers and clinicians in certain settings.

Accurate electrochemical detection of ROS in the brain is considerably challenging due to the presence of various molecules with similar oxidation/reduction potentials as the target ROS. This similarity can lead to false signaling, complicating the measurement process. To overcome this limitation, modifying the electrode with a selective membrane can prevent unwanted compounds from reaching the electrode surface. This approach can improve the accuracy of ROS measurements in the brain. In this regard, Wilson et al. developed a selective H₂O₂ biosensor by electrodeposition of a size exclusion membrane, 1,3-phenylenediamine (mPD), on a CFME [136]. This design addresses the issue of selective identification, as H₂O₂ shares similar voltammograms with other molecules, such as adenosine and histamine. Their biosensor enables real-time quantification of oxidative stress and its possible effects on DA dynamics in a single location in the brain using fast-scan cyclic voltammetry (FSCV) (Figure 4a). The study demonstrated the sensor’s ability to selectively detect H₂O₂ in the rat dorsal striatum, both in vitro and in vivo, and its stability over 4 h of use. The researchers also investigated the extracellular environment in rat brain tissue to evaluate the capacity of the mPD coating to ensure selective measurements of H₂O₂ in tissue. The biosensor successfully quantified H₂O₂ in intact brain tissue after 1 min local microinfusion of saline and MCS, validating its potential for in situ measurements in the brain (Figure 4b and c). However, FSCV involves the painstaking identification of the targeted signal from complex data and manipulation of the calibration to overcome signal drift in vivo.

Figure 4

In vivo ROS biosensors: (a) SEM of a carbon fiber surface after electrodeposition of the mPD polymer. (b) H₂O₂ quantification is simultaneously recorded on mPD-coated microelectrodes when MCS is microinfused in the brain tissue and the current is shown by color plots. (c) The quantified H₂O₂ concentration at different times obtained from (b), which shows the impact of local microinfusion of saline (black) and MCS (red) [136].

Luo et al. utilized another approach to decrease the interference effect in measuring H₂O₂ based on designing recognition molecules [137]. In their design, the H₂O₂ recognition sensor leverages the specific reaction between hydrogen peroxide and boric acid esters and, generates electrochemically active phenol and an electrochemical signal corresponding to the H₂O₂ level. To achieve this, the researchers used thioctic acid to synthesize 5-(1,2-dithiolan-3-yl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pentanamide (DBP), which served as an electrochemical probe for H₂O₂ recognition. To enhance in vivo detection accuracy, they incorporated electrochemically oxidized graphene oxide (EOGO) on the CFME as a built-in reference element. A pine branch-like gold (Au) nanostructure was then deposited on the CFME/EOGO electrode, and the DBP derivatives were attached through an Au–S bond, resulting in the construction of the electrochemical microsensor. In the presence of H₂O₂, DBP reacted to produce 5-(1,2-dithiolan-3-yl)-N-(4-hydroxyphen-yl) pentanamide (DHP) through a nucleophilic reaction, leading to increased detection current. The ratio of oxidation currents between DHP and EOGO allowed for accurate H₂O₂ detection. Ultimately, the electrochemical microsensor was effectively used for in vivo measurements of H₂O₂ in the brains of mice with PD, and the average H₂O₂ levels were systematically compared across different brain regions in these mice [137].

One of the biggest challenges for in vivo electrochemical measurement of ROS is the biofouling of electrode surfaces by small molecules and proteins. This greatly reduces the sensitivity, stability, and lifetime of the biosensor. Therefore, it is necessary to improve the anti-fouling performance of the electrode. To address this challenge, the Su group developed a low-fouling H₂O₂ electrochemical sensor, consisting of a silica nanomembrane (SNM) as an antifouling layer and Pt nanostructures as a catalyst [138]. The platinized silica nanoporous membrane (Pt@SNM) electrode displayed short-term antifouling performance in the in vivo environment, with sensitivity ratios of post- and pre-calibration curves calculated to be 47.3 and 20.0%, respectively. Also, a fast-current response to the injection of H₂O₂, with both the response time and response current remaining stable for up to 90 min, was observed [138].

5.2 Electrochemical measurement of superoxide

As mentioned earlier the determination of superoxide is harder compared to hydrogen peroxide due to its low stability and half-life. One of the few examples of the electrochemical determination of superoxide in the brain tissue is reported by Shim and coworkers. To construct a stable and sensitive microelectrode they first made the poly-5,2:5,2-terthiophene-3-carboxylic acid (poly-TTCA) film, which is a conductive polymer, on a needle-type gold microelectrode, followed by co-immobilization of lipid and Cytochrome c (Cyt c) on the Poly-TTCA film by covalent bonding (Figure 5a) [139]. After optimizing conditions, the electrode was implanted into a rat brain to probe the O₂ ^•– release in the extracellular space stimulated by acute and repeated administration of cocaine (Figure 5b). Amperometric results confirm the promising potential of the as-fabricated biosensor for in vivo measurement of superoxide in response to stimulant drug exposure (Figure 5c). In addition, the low applied potential required for amperometric detection in this design (−0.24 V) eliminates most of the interference for electrochemical detection of superoxides such as DA, uric acid, ascorbic acid, and glucose [139].

Figure 5

In vivo superoxide biosensors: (a) schematic representation of modifying an Au microelectrode (100 μm) using poly-TTCA, DGPD lipid, and cytochrome c to fabricate the micro-biosensor. (b) The photograph shows how the microelectrode was placed in a rat's brain during in vivo experiment. (c) The amperometric responses for O₂ ^•− concentration obtained by poly-TTCA/DGPD lipid/cytochrome c microelectrode implanted in the rat brain [139].

Another design was introduced by Peng et al. to design an enzyme-based electrochemical sensor for in vivo superoxide measurement in the brain [140]. The microsensor was designed by coating a functionalized ionic liquid polymer (PIL) onto PB nanoparticles (PBNPs) and CNTs. This design offers abundant interaction sites with SOD to prevent enzyme leakage from the sensor, enhancing sensitivity. The PIL itself possesses excellent biocompatibility, as demonstrated by cytotoxicity tests and bioactivity assays. The sensor demonstrated real-time measurement of O₂ ^•− release from living cells and in vivo biosensing of O₂ ^•− level changes in AD rat brains with high sensitivity and specificity. The modified sensor showed excellent stability, reproducibility, and repeatability toward O₂ ^•− monitoring. However, the sensitivity of the sensor decreased by approximately 60% after implantation in the brain due to the “fouling” of the sensor surface. Despite this decrease, the sensitivity was still high enough to monitor O₂ ^•− variations in vivo.

In another study, Huang et al. utilized diphenylphosphonate-2-naphthol ester (ND) as a specific recognition molecule for the selective determination of O₂ ^•– [141]. The sensor also included an inner reference of methylene blue (MB) co-assembled at the electrode surface to enhance the determination accuracy of O₂ ^•–. The anodic peak current ratio between 2-naphthol and MB showed a linear relationship with the concentration of O₂ ^•–, allowing for accurate detection of O₂ ^•– concentrations from 2 to 200 μM. The electrochemical sensor demonstrated high selectivity towards O₂ ^•– detection against various potential interferences in the brain, as well as good stability even after storage for 7 days. The sensor was successfully applied to monitor O₂ ^•– levels in the rat brain, specifically in the hippocampus, cortex, and striatum regions, during cerebral ischemia. The results showed that the levels of O₂ ^•– increased in these regions during cerebral ischemia, and this increase was more pronounced in diabetic rats compared to normal rats. The non-enzymatic electrochemical sensor developed in this study showed excellent selectivity, stability, and accuracy in detecting O₂ ^•– concentrations in the rat brain during cerebral ischemia. The study suggests that this sensor has the potential for use in exploring the roles of O₂ ^•– in brain functions and could be extended to develop other sensors for the in vivo analysis of metal ions, ROS, and more in living systems. However, the detection range of the sensor was also limited to a concentration range of 2–200 μM, which may not be sufficient for detecting very low or very high levels of O₂ ^•– in some biological samples. Additionally, the sensor was only tested in rats and may not apply to other animal models or human subjects without further validation. Finally, the in vivo experiments were conducted under controlled laboratory conditions, and it remains to be seen how the sensor would perform in more complex and dynamic environments, such as during spontaneous seizures or under conditions of acute stress [141].

5.3 Comparative evaluation of electrochemical sensor designs for ROS detection in the brain

Several designs have been discussed for in vivo measuring ROS in the brain using electrochemical sensors. The electrode designs included PB-based biosensors, ring-disk microelectrodes, ratiometric biosensors, size-exclusion membrane-based sensors, and enzyme-based sensors. Each of these designs has its unique advantages and disadvantages (Table 2).

Table 2

Benchmark of different electrochemical biosensor designs for in vivo ROS measurement in the brain

Electrode design	Electrochemical technique	Advantage	Disadvantage	Ref.
PB-CNT/PDA-CFME	Amperometry	Excellent selectivity, interference-free, good stability	Long-term stability and performance not addressed, fabrication scalability and reproducibility	[133]
PB-PEDOT-CFME/Au-ring electrode	Amperometry	Simultaneous detection of H₂O₂ and DA, sensitive and selective	Simultaneous changes in H₂O₂ and DA during electrical stimulation were not observed	[134]
Cat/Fc/SWNT/CFME	Cyclic voltammograms	Simultaneous detection of H₂O₂ and pH, high sensitivity, linearity, and selectivity	Invasive implantation, sensor accuracy affected by the surrounding environment, stability over time	[135]
mPD-CFME	FSCV	Highly selective for H₂O₂, and good stability over 4 h of use	Complex data analysis, and calibration to overcome signal drift in vivo	[136]
DBP/EOGO/CFME	Amperometry	Recognition molecule-based selectivity, built-in reference element	Invasive implantation, sensor accuracy affected by the surrounding environment, stability over time	[137]
Pt@SNM	Amperometry	Low-fouling, short-term antifouling performance, stable response up to 90 min	Limited long-term antifouling performance	[138]
Poly-TTCA/Cyt c	Amperometry	Low applied potential, eliminates most interference, sensitive detection of superoxide	Limited examples for in vivo measurement of superoxide	[139]
PIL-PBNPs-CNT/SOD	Amperometry	Sensitive and specific detection of superoxide, excellent stability, and reproducibility	Sensitivity decreased by 60% after implantation due to sensor fouling	[140]

Prussian, blue-based biosensors show excellent selectivity for H₂O₂ detection and are interference-free from O₂ and other electroactive species. However, their long-term stability and performance in vivo need further investigation. Ring-disk microelectrodes allow simultaneous detection of H₂O₂ and neurotransmitters like DA, providing a more comprehensive understanding of the brain’s neurophysiology and pathology. However, further improvements in selectivity and sensitivity are necessary for more accurate detection and monitoring of multiple substances simultaneously. The ratiometric design also offers high sensitivity, linearity, and selectivity but may be affected by the surrounding microenvironment and the stability of the sensor over time. Size-exclusion membrane-based sensors, such as the one developed by Wilson et al., can selectively detect H₂O₂ in the brain using FSCV. However, the identification of the targeted signal from complex data remains a challenge for this technique. Enzyme-based sensors can detect superoxide in the brain with high sensitivity and specificity but may face issues with fouling and reduced sensitivity after implantation. Overall, while each design has its merits, there is no one-size-fits-all solution for ROS measurement in the brain. Further research is needed to improve the stability, selectivity, and sensitivity of these designs to accurately measure ROS in vivo and advance our understanding of the role of ROS in various neurological disorders and their progression to get a better insight into different approaches. When comparing various methods for in vivo electrochemical detection of ROS in the brain, it is clear that each approach has managed to tackle some of the inherent obstacles linked to this technique. Nevertheless, there is still potential for enhancement by studying in vivo electrochemical strategies utilized in distinct tissues and drawing insights from their achievements and limitations. Moreover, it is crucial to address the inherent invasiveness of the electrochemical method, which may cause tissue damage and impact the accuracy of the measurements.

One potential solution to decrease the invasiveness of these measurements is employing micro- or nano-electrodes. These smaller-sized electrodes can potentially inflict less tissue damage while retaining sensitivity and selectivity. Furthermore, enhancing the physical flexibility of the electrodes could further diminish the invasiveness of the method. Flexible electrodes can adapt to the tissue surface, mitigating mechanical stress and minimizing possible damage to nearby cells and tissues. Additionally, progress in electrode surface modification methods and materials can lead to improvements in the sensitivity and selectivity of the sensors. For example, the use of nanomaterials or biocompatible coatings can bolster the sensor’s performance while minimizing any detrimental effects on the surrounding tissues.

Overall, although current designs for in vivo electrochemical detection of ROS in the brain have made substantial progress in addressing the technique’s challenges, there is still scope for further improvement. By investigating successful approaches in other tissues, optimizing electrode size and flexibility, and employing cutting-edge surface modification techniques, we can potentially develop more accurate, sensitive, and minimally invasive electrochemical sensors for ROS detection in the brain.

6 Electrochemical measurement of RNS in the brain

RNS are essential signaling molecules that play a crucial role in various physiological processes, particularly in the nervous system. RNS contribute to critical functions such as the regulation of blood flow, synaptic plasticity, and neurotransmission. Moreover, they are involved in modulating immune responses and maintaining cellular homeostasis. Despite their beneficial effects, an imbalance in RNS production can lead to detrimental outcomes. Excessive RNS levels can result in nitrosative stress, causing oxidative damage to proteins, lipids, and DNA, ultimately impairing neuronal function. This damage has been linked to the development and progression of various neurodegenerative diseases, such as AD, PD, and multiple sclerosis. Therefore, understanding the physiological roles and maintaining the balance of RNS in the nervous system is crucial for preserving neuronal health and preventing neurological disorders. The two most important and well-documented RNS are nitric oxide and ONOO⁻, and thus in this section we mainly focus on their in vivo quantification in the brain using electrochemical sensors.

6.1 Electrochemical measurement of nitric oxide in the brain

Nitric oxide (NO) is the most extensively studied RNS, especially in electrochemical systems. To fabricate a biosensor for in vivo determination of NO one should take into account (i) sensitivity: down to the nanomolar range, (ii) selectivity factors: higher than 100 against the other species, (iii) fast response time: in the range of millisecond, (iv) stability: higher than 2 h, (v) small size of electrode: in the range of 10–50 μm, and (vi) ease of handling [142].

From an electrochemical standpoint, NO can undergo a one-electron reduction under cathodic conditions, with a potential between −0.5 and −1.4 V (vs Ag/AgCl), depending on the electrode type and pH. Alternatively, it can experience three-electron oxidation, typically at 0.8 V (vs Ag/AgCl). However, to eliminate oxygen interference in the electrochemical detection of NO, most studies – particularly those focusing on monitoring brain oxidative stress – emphasize the electrooxidation of NO [123].

Meiller et al. developed a highly selective microsensor using a fluorinated xerogel coating for in vivo detection of NO in the brain [143]. The microsensor, consisting of a CFME covered with nickel porphyrin and the fluorinated xerogel, was able to detect NO released in response to N-methyl-d-aspartate (NMDA) injection in the rat parietal cortex. The in vivo recordings demonstrated the ability of the microsensor to provide precise measurements of NO levels, despite the electrical noise related to ongoing neuronal activity around the microsensor. However, the study also found interindividual variability in the cortical response to NMDA microinjection, and the sensitivity of the microsensor was limited to concentrations above 166 ± 15 nM in the cortex. The use of the fluorinated xerogel coating in the microsensor offers superior selectivity compared to other types of screening layers such as Nafion, reducing unwanted oxidation of endogenous redox molecules. This makes the microsensor suitable for investigating endogenous NO release in vivo in both physiological and pathological conditions and should improve the accuracy of future brain NO monitoring. However, the study’s findings are limited to the use of the microsensor in response to NMDA microinjection in the rat parietal cortex, and the sensitivity of the microsensor is limited to concentrations above 166 ± 15 nM in the cortex. Further research is needed to assess the applicability of the microsensor to other brain regions and experimental conditions.

Another incorporation of fluorinated xerogel coating was used by the Suh group to develop an electrochemical biosensor for simultaneous measurement of NO and carbon monoxide (CO) [144]. In this design, a dual microelectrode system including an Au-deposited Pt microdisk (WE1) which can selectively detect CO (at 0.2 V vs Ag/AgCl), and a Pt black-deposited Pt disk (WE2) that can be used for selective anodic detection of NO (+0.75 V vs Ag/AgCl). To provide exclusive selectivity toward the biological interferences and improve response time, the electrodes were coated with fluorinated xerogel (Figure 6a). Also, the miniaturized size (end plane diameter of less than 300 μm) as well as tapered needle-like geometry facilitated the insertion of the sensor into biological tissues. To explore the applicability of the sensor for simultaneous measurement of NO and CO, it was placed in the cortical tissue of a living rat brain, and 4-aminopyridine was used to induce acute seizure conditions (Figure 6b). The activity of the sensor in cortical tissue measurement exhibited well-defined NO and CO changes which could be characterized by (i) alteration in peak-shaped, (ii) sustaining steady-state level, and (iii) slow decline back to the background levels (Figure 6c) [144].

Figure 6

In vivo RNS biosensor. (a) schematic representation of fabrication steps for an insertable microsensor for NO and CO measurement. (b) The experimental setup for quantification of nitric oxide and carbon monoxide using as-fabricated microelectrode inserted into cortical layer after epileptic seizures induced by injecting 15 mM of aminopyridine. (c) The signal changes of local field potential, nitric oxide, and carbon monoxide in response to epileptic seizures [144]. (d) The overall illustration of a system designed for simultaneously monitoring nitric oxide and oxygen. The black box on the right side demonstrated the exposed brain area in this experiment. (e) A typical response of the designed microsensor to changes in oxygen and nitric oxide levels. (f) The change in oxygen level (∆C_O2, norm, 0) and corresponding normalized nitroxide level changes (∆C_NO, norm, 9) measured in a different animal using NO/O₂ microsensor [147]. (g) The experimental setup for measuring nitric oxide using a scanning electrochemical microscope (SECM). In this design, the SECM is set for a two-dimensional scan at a constant z-direction, 10 μm spacing in the y-direction, and a moving rate of 5 μm s⁻¹ in the x-direction. The rightmost insert demonstrates a nanopore fabricated by a platinized NO sensor. (h) A three-dimensional demonstration of the measured NO level alongside the immunohistochemical analysis data [148]. (i) The corresponding color plot (e vs i vs time) obtained by square wave voltammograms in response to the different exogenous levels of NO in the rat hippocampus [149].

Suh’s group further proceeded to test the effectiveness of their microsensor design by using it to measure real-time levels of NO and CO in different regions of the brain during acute seizures [145]. The study found that seizures that spread through cortical layers to adjacent areas in the brain resulted in different changes in NO and CO concentrations depending on their relative location to the seizure focus. The high spatiotemporal NO/CO dual microsensor was able to accurately measure the intimate connection between NO and CO in seizure events with high sensitivity and selectivity.

In another study, the NO and potassium ions (K⁺) were simultaneously measured. In this study, a microsensor was developed to detect both nitric oxide (NO) and potassium ions (K⁺) simultaneously in biological tissues [146]. The sensor had a needle-like geometry and was composed of a Pt-coated electrode modified with fluorinated xerogel for NO detection (WE1) and an Ag-coated electrode loaded with a K⁺ ion-selective membrane for K⁺ detection (WE2). WE1 operated in amperometric mode, measuring current in response to NO concentration changes, while WE2 operated in potentiometric mode, measuring potential changes in response to K⁺ concentration changes. The sensor demonstrated high sensitivity and selectivity with WE1 and WE2 sensing NO and K⁺, respectively, without crosstalk between their signals. The sensor was successfully applied to monitor NO and K⁺ changes in a living rat brain cortex during induced epileptic seizures, with changes in NO and K⁺ levels showing a clear correlation with electrophysiological recordings of seizures. The sensor’s sensitivity and response time were sufficient to measure dynamic changes in NO and K⁺ levels during seizures, and the sensor geometry allowed for its insertion into deep cortical layers. The study suggests that the dual sensor could have potential applications in basic research areas requiring concurrent real-time measurements of NO and K⁺ [146].

Park et al. also developed an electrochemical sensor for the real-time monitoring of the endogenous NO release and oxygen consumption profile in a rat neocortex, using the amperometric technique (Figure 6d) [147]. In this design, two microelectrodes were used as working electrodes (WEs), one for NO and the other for O₂ monitoring. Both working microelectrodes were fabricated using a platinized Pt disk which was covered by the PTFE gas-permeable membrane. The dynamical changes of nitric oxide and oxygen were probed using amperometry while applying an electrical stimulation (25 s with 1 mA amplitude and 0.45 Hz) that could trigger transient cerebral hypoxia by increasing metabolic demands inside the stimulated cortical region (Figure 6e).

The dual sensor could quantitatively measure the dynamic relationship between oxygen level in tissue and released nitric oxide with sufficient temporal resolution. This design could detect a 3.6 (±0.9)-fold increase in the nitric oxide concentration and a 0.41 (±0.04)-fold decrease in oxygen concentration upon electrical stimulation of the cortical region. Eventually, an assessment of 11 rats showed that there is a strong relation between tissue oxygenation, cerebral metabolic demands, and the level of nitric oxide released for vasodilation induced by direct electrical stimulation (Figure 6f) [147].

Apart from conventional electrochemical techniques, SECM can be used for two-dimensional imaging of the local analyte concentration. The first report on using SECM for real-time in vivo nitric oxide imaging is reported by Jo et al. by coupling an amperometric NO sensor with SECM (Figure 6g) [148]. In this technique, a microelectrode is utilized to measure the current as a function of the electrode position while simultaneously scanning across the sample surface in close proximity. A planar-type nitric oxide nanoelectrode was used as the probe tip in SECM, in order to measure the NO release dynamic regarding the location of samples (in two-dimensional geometry). Besides the in vivo two-dimensional feature, this design could provide information on the real-time distribution of NO in an intact living brain in a three-dimensional space with spatial resolution and high sensitivity (Figure 6h). Thus, the local concentration of NO at the cortical surface of a living mouse brain was monitored by electrochemical amperometric, and the change in the current related to the NO level on a nanometer-sized sensing electrode was projected as a graphical image representing the NO spatial level. However, this technique suffers from (i) a relatively long data acquisition time (e.g., ∼100 min), and (ii) restricted to surface scanning [148].

As explained earlier in this review, most of the NO sensors are designed based on the electrooxidation of NO to eliminate oxygen interference in measurement. One of the rare examples of the nitric oxide electrochemical measurement in the brain based on the NO reduction is introduced by Barbosa and coworkers. In this design, first, a mixture of hemin and multi-wall CNT was covalently cross-linked to the chitosan, thus the CNT could improve the sensitivity of the electrode [150,151,152,153]. Then, p-benzoquinone was added to the mixture and electrodeposition was used to deposit the mixture on the carbon fiber surface. In fact, the reduction of p-benzoquinone during electrodeposition at −0.5 V (vs Ag/AgCl) increases the local pH by consuming protons at the microelectrode surface, which results in chitosan insolubilization and attachment to the surface of carbon fiber. The as-fabricated microsensors were implanted into the rat hippocampus enabling in vivo assessment of exogenous NO applied locally (Figure 6i) [149].

Overall, in this section, we discussed various studies on electrochemical sensors for the detection of nitric oxide (NO). While these sensors offer some advantages, they also have certain limitations, such as their invasive nature and complexity, which can hinder real-time, online monitoring applications. To address these challenges, researchers have explored new materials strategies, device architectures, and fabrication schemes to develop flexible, degradable electrochemical sensors for real-time NO detection. One example is the work by Yin et al., where they demonstrated a sensor with a low detection limit (3.97 nM), a wide sensing range (0.01–100 μM), a high temporal resolution (<350 ms), and desirable anti-interference characteristics [154]. This sensor is capable of real-time monitoring not only at the cellular and organ levels but also in the joint cavity of a rabbit for 5 days with a wireless data transmission system.

This device is engineered for total physical transience, both in vitro and in vivo, utilizing potential hydrolysis, breakdown, phagocytosis, and metabolic clearance mechanisms. Evaluations of biocompatibility reveal no considerable negative consequences or build-up of foreign substances at implantation locations or within vital organs. The method for detecting NO relies on amperometry utilizing a conventional three-electrode setup, with gold nanomembranes functioning as the WE, counter electrode, and reference electrode (RE). The redox reaction involves the oxidation of a molecule of NO to nitrosonium ion (NO⁺) on the surface of the WE, followed by a subsequent conversion to nitrite (NO²⁻) in the solution. The performance of the fabricated NO sensors is evaluated at 37°C. Linear sweep voltammetry measurements reveal an oxidation potential of approximately 0.8 V (WE vs RE) in the presence of NO. Time-dependent current response measurements at different NO concentrations demonstrate rapid current response capture (<350 ms), important for real-time NO monitoring. Calibration curves show a linear relationship between the NO concentration and the response current, with a detection limit of 3.97 nM [154]. Eventually, although the study primarily performed ex vivo analysis on rat brain tissue, in vivo analysis of a rat’s heart showed promising potential for possible in vivo measurements in other organs, such as the brain. These advances in flexible and biodegradable NO sensing with accurate and stable characteristics in physiological conditions offer essential diagnostic and therapeutic information, overcoming some of the limitations of traditional electrochemical sensors.

6.2 Electrochemical measurement of peroxynitrite in the brain

Peroxynitrite (ONOO⁻) is a highly reactive RNS molecule that can play both protective and deleterious roles in cells and organisms. While it has bactericidal effects, ONOO⁻ can also cause cell death through the oxidation of lipids, proteins, and DNA. Abnormal levels of ONOO⁻ have been linked to various human diseases, such as acute liver injury, epilepsy, inflammation, neurodegenerative disorders, stroke, and drug-induced acute kidney injury, among others. Therefore, developing highly sensitive and selective tools for the ONOO⁻ detection in biological systems is crucial.

However, only a few electrochemical studies have been conducted on in vivo peroxynitrite measurement in the brain, primarily due to its short half-life (0.9 s at pH 7.4) and intrinsic high reactivity. Liu et al. developed a ratiometric electrochemical biosensor for real-time monitoring and determination of ONOO⁻ in the rat brain during cerebral ischemia [155]. In this design, they first deposited gold nanoleaves on a carbon fiber electrode, then modified the as-prepared electrode by an organic molecule conjugated to ferrocene as an electroactive group (Figure 7a and b). The ferrocene oxidation peak decreased by increasing the ONOO⁻ concentration, providing a selective sensor for peroxynitrite, with a fast response within 15 s. To avoid the environmental effects, they used 5′-MB-GGCGCGATTTT-SH-3′ (SH-DNA-MB) as an inner reference molecule. Thus, the oxidation peak current ratio between MB and ferrocene was utilized for the analytical measurement of peroxynitrite. The developed biosensor was selective against amino acids, bioactive species, and metal ions which are the main potential interferences in the brain matrix. Further validation was done by implanting micro-biosensors in different regions of the rat brain including the hippocampus, striatum, and cortex regions for real-time monitoring of ONOO⁻ (Figure 7c) [155].

Figure 7

Real-time monitoring of ONOO⁻ using a ratiometric electrochemical biosensor. (a) The schematic representation of electrochemical biosensor design, (b) the SEM images of CFME electrodeposited with Au nanoleaves, and (c) levels of ONOO⁻ determined using as-fabricated electrodes in the cortex, striatum, and hippocampus regions of rat brains followed by global cerebral ischemia at different times [155].

Although there is limited research on the electrochemical detection of peroxynitrite in the brain, the promising results obtained from other tissues or live cells suggest that these sensors could potentially be applied to peroxynitrite measurement in the brain. The development of electrochemical sensors for ONOO⁻ detection may provide essential diagnostic and therapeutic information for various neurological diseases and conditions, such as AD, PD, multiple sclerosis, and TBIs, where RNS dysregulation has been implicated. To advance the field of peroxynitrite sensing in the brain, it is important to address the challenges associated with the short half-life and high reactivity of ONOO⁻. This could involve the development of new materials or surface modifications that can enhance the stability and selectivity of the sensors. Additionally, the integration of wireless and real-time monitoring capabilities into these sensors could facilitate continuous tracking of ONOO⁻ levels in living animals, providing valuable insights into disease progression and treatment efficacy.

7 Outlook and conclusion

ROS/RNS are crucial for normal physiological functions, but when present in excess, they can cause oxidative harm to DNA, proteins, and lipids, leading to numerous disorders, particularly those involving the brain and nervous systems. Monitoring ROS/RNS levels in real-time during different neurochemical events has long been a challenging task for scientists studying the brain’s chemical activities. Electrochemical systems employing micro- and nanoelectrodes, created using nano-engineering techniques, demonstrate considerable promise for in vivo ROS/RNS tracking due to benefits such as high sensitivity and low detection thresholds, excellent selectivity, compatibility with in vivo measurements because of their ultra-small size, and affordability.

Nonetheless, many of these methods encounter difficulties concerning large-scale applicability, reproducibility, accuracy, and the presence of interference. To tackle these problems, researchers can investigate nanoelectrodes augmented with nanomaterials and nanocomposites that provide low impedance and increased stability, leading to enhanced temporal and spatial resolution. Moreover, integrating electrochemistry with other analytical tools, using micro-electromechanical systems, or adopting 3D printing and lithography techniques could result in highly accurate and reproducible miniaturized micro- and nanoelectrodes.

As we move forward, the development of the next generation of in vivo sensors requires addressing several critical challenges. For instance, identifying molecules with similar functional groups and molecular structures, including neurotransmitters, amino acids, and proteins, is still challenging. Additionally, the rapid biological releases of active molecules demand that the sensors have a fast response time. Furthermore, electrode fouling due to the complexity of the matrix and the adsorption of different biological entities such as protein and fatty acids are still challenges for the long-term application of these biosensors. Eventually, the invasive features of these types of sensors need to be addressed.

To tackle these hurdles, researchers could investigate developing supramolecular probes with multi-site recognition abilities based on a variety of chemical bonding interactions. This strategy would allow for the highly selective identification of complex biomolecules. Additionally, innovative technologies such as computer-aided molecular simulation design and thermodynamic simulation calculations could guide advancing in vivo sensors with intricate molecular recognition and rapid response capabilities. Progress in microfabrication and lithography might also improve the channel density of electrochemical microelectrode arrays, facilitating the recording of chemical signal networks across multiple brain regions. By combining in vivo electrochemical techniques with fiber-optic photometry, the real-time distribution of chemical signals throughout the brain can be mapped, yielding invaluable insights for understanding brain physiological and pathological processes as well as drug screening. Ultimately, researchers in the field of ROS/RNS recognition in the brain could benefit from other cutting-edge designs implemented in various biological organs and/or cells, including but not limited to cancer and tumor cells, liver, heart, and more [156,157,158].

Funding information: This work was supported by the Korea government (MSIT) (2022M3J7A1062940).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Conflict of interest: The authors state no conflict of interest.

References

[1] Babcock GT. How oxygen is activated and reduced in respiration. Proc Natl Acad Sci. 1999;96(23):12971–3.10.1073/pnas.96.23.12971Search in Google Scholar PubMed PubMed Central

[2] Halliwell B. Reactive oxygen species and the central nervous system. J Neurochem. 1992;59(5):1609–23.10.1111/j.1471-4159.1992.tb10990.xSearch in Google Scholar PubMed

[3] Yuasa M, Oyaizu K. Electrochemical detection and sensing of reactive oxygen species. Curr Org Chem. 2005;9(16):1685–97.10.2174/138527205774610921Search in Google Scholar

[4] Salim S. Oxidative stress and the central nervous system. J Pharmacol Exp Ther. 2017;360(1):201–5.10.1124/jpet.116.237503Search in Google Scholar PubMed PubMed Central

[5] Di Meo S, Reed TT, Venditti P, Victor VM. Role of ROS and RNS sources in physiological and pathological conditions. Oxid Med Cell Longev. 2016;2016:1–44.10.1155/2016/1245049Search in Google Scholar PubMed PubMed Central

[6] Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24(10):R453–62.10.1016/j.cub.2014.03.034Search in Google Scholar PubMed PubMed Central

[7] Mittler R. ROS are good. Trends Plant Sci. 2017;22(1):11–9.10.1016/j.tplants.2016.08.002Search in Google Scholar PubMed

[8] Mittal C, Murad F. Guanylate cyclase: Regulation of cyclic GMP metabolism. Cycl Nucleotides: Part I: Biochem. 1982;24:225–60.10.1007/978-3-642-68111-0_5Search in Google Scholar

[9] Schreck R, Albermann K, Baeuerle PA. Nuclear factor kB: An oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun. 1992;17(4):221–37.10.3109/10715769209079515Search in Google Scholar PubMed

[10] Di Marzo N, Chisci E, Giovannoni R. The role of hydrogen peroxide in redox-dependent signaling: homeostatic and pathological responses in mammalian cells. Cells. 2018;7(10):156.10.3390/cells7100156Search in Google Scholar PubMed PubMed Central

[11] Rendra E, Riabov V, Mossel DM, Sevastyanova T, Harmsen MC, Kzhyshkowska J. Reactive oxygen species (ROS) in macrophage activation and function in diabetes. Immunobiology. 2019;224(2):242–53.10.1016/j.imbio.2018.11.010Search in Google Scholar PubMed

[12] Yang Y, Wang Y, Guo L, Gao W, Tang T-L, Yan M. Interaction between macrophages and ferroptosis. Cell Death Dis. 2022;13(4):355.10.1038/s41419-022-04775-zSearch in Google Scholar PubMed PubMed Central

[13] Herb M, Schramm M. Functions of ROS in macrophages and antimicrobial immunity. Antioxidants. 2021;10:313. s Note: MDPI stays neutral with regard to jurisdic-tional claims in …; 2021.10.3390/antiox10020313Search in Google Scholar PubMed PubMed Central

[14] Popa-Wagner A, Mitran S, Sivanesan S, Chang E, Buga A-M. ROS and brain diseases: The good, the bad, and the ugly. Oxid Med Cell Longev. 2013;2013:1–14.10.1155/2013/963520Search in Google Scholar PubMed PubMed Central

[15] Simpson DS, Oliver PL. ROS generation in microglia: understanding oxidative stress and inflammation in neurodegenerative disease. Antioxidants. 2020;9(8):743.10.3390/antiox9080743Search in Google Scholar PubMed PubMed Central

[16] Cobley JN. Synapse pruning: mitochondrial ROS with their hands on the shears. Bioessays. 2018;40(7):1800031.10.1002/bies.201800031Search in Google Scholar PubMed

[17] Beckhauser TF, Francis-Oliveira J, De, Pasquale R. Reactive oxygen species: physiological and physiopathological effects on synaptic plasticity: Supplementary issue: Brain plasticity and repair. J Exp Neurosci. 2016;10:JEN. S39887.10.4137/JEN.S39887Search in Google Scholar PubMed PubMed Central

[18] Stefanatos R, Sanz A. The role of mitochondrial ROS in the aging brain. FEBS Lett. 2018;592(5):743–58.10.1002/1873-3468.12902Search in Google Scholar PubMed

[19] Oswald MC, Garnham N, Sweeney ST, Landgraf M. Regulation of neuronal development and function by ROS. FEBS Lett. 2018;592(5):679–91.10.1002/1873-3468.12972Search in Google Scholar PubMed PubMed Central

[20] Campos PB, Paulsen BS, Rehen SK. Accelerating neuronal aging in in vitro model brain disorders: A focus on reactive oxygen species. Front Aging Neurosci. 2014;6:292.10.3389/fnagi.2014.00292Search in Google Scholar PubMed PubMed Central

[21] Newsholme P, Homem De Bittencourt Jr PI, O’Hagan C, De Vito G, Murphy C, Krause MS. Exercise and possible molecular mechanisms of protection from vascular disease and diabetes: the central role of ROS and nitric oxide. Clin Sci. 2010;118(5):341–9.10.1042/CS20090433Search in Google Scholar PubMed

[22] Guo JD, Zhao X, Li Y, Li GR, Liu XL. Damage to dopaminergic neurons by oxidative stress in Parkinson’s disease. Int J Mol Med. 2018;41(4):1817–25.10.3892/ijmm.2018.3406Search in Google Scholar PubMed

[23] Shohami E, Kohen R. The role of reactive oxygen species in The Pathogenesis of Traumatic Brain Injury. In: Gadoth N, Göbel H, eidtors. Oxidative stress and free radical damage in Neurology. Humana Press; 2011. p. 99–118.10.1007/978-1-60327-514-9_7Search in Google Scholar

[24] Bandyopadhyay U, Das D, Banerjee RK. Reactive oxygen species: Oxidative damage and pathogenesis. Curr Sci. 1999;77:658–66.Search in Google Scholar

[25] Boraei SBA, Nourmohammadi J, Mahdavi FS, Zare Y, Rhee KY, Montero AF, et al. Osteogenesis capability of three-dimensionally printed poly (lactic acid)-halloysite nanotube scaffolds containing strontium ranelate. Nanotechnol Rev. 2022;11(1):1901–10.10.1515/ntrev-2022-0113Search in Google Scholar

[26] Murphy MP, Bayir H, Belousov V, Chang CJ, Davies KJ, Davies MJ, et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 2022;4(6):651–62.10.1038/s42255-022-00591-zSearch in Google Scholar PubMed PubMed Central

[27] Calas-Blanchard C, Catanante G, Noguer T. Electrochemical sensor and biosensor strategies for ROS/RNS detection in biological systems. Electroanalysis. 2014;26(6):1277–86.10.1002/elan.201400083Search in Google Scholar

[28] Galadari S, Rahman A, Pallichankandy S, Thayyullathil F. Reactive oxygen species and cancer paradox: to promote or to suppress? Free Radic Biol Med. 2017;104:144–64.10.1016/j.freeradbiomed.2017.01.004Search in Google Scholar PubMed

[29] Niedzielska E, Smaga I, Gawlik M, Moniczewski A, Stankowicz P, Pera J, et al. Oxidative stress in neurodegenerative diseases. Mol Neurobiol. 2016;53:4094–125.10.1007/s12035-015-9337-5Search in Google Scholar PubMed PubMed Central

[30] Newsholme P, Cruzat VF, Keane KN, Carlessi R, de Bittencourt PIH Jr. Molecular mechanisms of ROS production and oxidative stress in diabetes. Biochem J. 2016;473(24):4527–50.10.1042/BCJ20160503CSearch in Google Scholar PubMed

[31] Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, et al. ROS and ROS-mediated cellular signaling. Oxid Med Cell Longev. 2016;2016:1–18.10.1155/2016/4350965Search in Google Scholar PubMed PubMed Central

[32] Angelova PR, Abramov AY. Functional role of mitochondrial reactive oxygen species in physiology. Free Radic Biol Med. 2016;100:81–5.10.1016/j.freeradbiomed.2016.06.005Search in Google Scholar PubMed

[33] Milatovic D, Zaja-Milatovic S, Gupta RC. Oxidative stress and excitotoxicity: Antioxidants from nutraceuticals. Nutraceuticals. Netherlands: Elsevier; 2016. p. 401–13.10.1016/B978-0-12-802147-7.00029-2Search in Google Scholar

[34] Vašková J, Vaško L. Introductory chapter: Unregulated mitochondrial production of reactive oxygen species in testing the biological activity of compounds. Medicinal chemistry. UK: IntechOpen London; 2018.10.5772/intechopen.82514Search in Google Scholar

[35] Aguilar TAF, Navarro BCH, Pérez JAM. Endogenous antioxidants: A review of their role in oxidative stress. A master regulator of oxidative stress-the transcription factor nrf2. England: IntechOpen; 2016. p. 3–20.10.5772/65715Search in Google Scholar

[36] Nimse SB, Pal D. Free radicals, natural antioxidants, and their reaction mechanisms. RSC Adv. 2015;5(35):27986–8006.10.1039/C4RA13315CSearch in Google Scholar

[37] Tarafdar A, Pula G. The role of NADPH oxidases and oxidative stress in neurodegenerative disorders. Int J Mol Sci. 2018;19(12):3824.10.3390/ijms19123824Search in Google Scholar PubMed PubMed Central

[38] Choi BY, Kim JH, Kho AR, Kim IY, Lee SH, Lee BE, et al. Inhibition of NADPH oxidase activation reduces EAE-induced white matter damage in mice. J Neuroinflammation. 2015;12(1):1–15.10.1186/s12974-015-0325-5Search in Google Scholar PubMed PubMed Central

[39] Kim JH, Jang BG, Choi BY, Kim HS, Sohn M, Chung TN, et al. Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death. Brain Res. 2013;1499:163–72.10.1016/j.brainres.2013.01.007Search in Google Scholar PubMed

[40] Kho AR, Choi BY, Lee SH, Hong DK, Lee SH, Jeong JH, et al. Effects of protocatechuic acid (PCA) on global cerebral ischemia-induced hippocampal neuronal death. Int J Mol Sci. 2018;19(5):1420.10.3390/ijms19051420Search in Google Scholar PubMed PubMed Central

[41] Lee SH, Choi BY, Kho AR, Jeong JH, Hong DK, Lee SH, et al. Protective effects of protocatechuic acid on seizure-induced neuronal death. Int J Mol Sci. 2018;19(1):187.10.3390/ijms19010187Search in Google Scholar PubMed PubMed Central

[42] Ma MW, Wang J, Dhandapani KM, Wang R, Brann DW. NADPH oxidases in traumatic brain injury–promising therapeutic targets? Redox Biol. 2018;16:285–93.10.1016/j.redox.2018.03.005Search in Google Scholar PubMed PubMed Central

[43] Ma MW, Wang J, Zhang Q, Wang R, Dhandapani KM, Vadlamudi RK, et al. NADPH oxidase in brain injury and neurodegenerative disorders. Mol Neurodegener. 2017;12:1–28.10.1186/s13024-017-0150-7Search in Google Scholar PubMed PubMed Central

[44] Lee SH, Lee M, Ko DG, Choi BY, Suh SW. The role of NADPH oxidase in neuronal death and neurogenesis after acute neurological disorders. Antioxidants. 2021;10(5):739.10.3390/antiox10050739Search in Google Scholar PubMed PubMed Central

[45] Unsal V, Dalkıran T, Çiçek M, Kölükçü E. The role of natural antioxidants against reactive oxygen species produced by cadmium toxicity: A review. Adv Pharm Bull. 2020;10(2):184.10.34172/apb.2020.023Search in Google Scholar PubMed PubMed Central

[46] Dao V, Altenhöfer S, Elbatreek M, Casas A, Lijnen P, Meens M, et al. Isoform-specific NADPH oxidase inhibition for pharmacological target validation. bioRxiv. 2018;1:382226.10.1101/382226Search in Google Scholar

[47] Park H-R, Oh R, Wagner P, Panganiban R, Lu Q. New insights into cellular stress responses to environmental metal toxicants. Int Rev Cell Mol Biol. 2017;331:55–82.10.1016/bs.ircmb.2016.10.001Search in Google Scholar PubMed

[48] de Araújo RF, Martins DBG, Borba MAC. Oxidative stress and disease. A master regulator of oxidative stress-the transcription factor nrf2. England: IntechOpen; 2016.10.5772/65366Search in Google Scholar

[49] Huang WJ, Zhang X, Chen WW. Role of oxidative stress in Alzheimer’s disease. Biomed Rep. 2016;4(5):519–22.10.3892/br.2016.630Search in Google Scholar PubMed PubMed Central

[50] Davies MJ. Protein oxidation and peroxidation. Biochem J. 2016;473(7):805–25.10.1042/BJ20151227Search in Google Scholar PubMed PubMed Central

[51] Qin Y. Health benefits of bioactive seaweed substances. Bioactive seaweeds for food applications. Netherlands; Elsevier; 2018. p. 179–200.10.1016/B978-0-12-813312-5.00009-1Search in Google Scholar

[52] Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J. 2015;15(1):1–22.10.1186/s12937-016-0186-5Search in Google Scholar PubMed PubMed Central

[53] Schönfeld P, Reiser G. Why does brain metabolism not favor burning of fatty acids to provide energy? -Reflections on disadvantages of the use of free fatty acids as fuel for brain. J Cereb Blood Flow Metab. 2013;33(10):1493–9.10.1038/jcbfm.2013.128Search in Google Scholar PubMed PubMed Central

[54] Auestad N, Korsak RA, Morrow JW, Edmond J. Fatty acid oxidation and ketogenesis by astrocytes in primary culture. J Neurochem. 1991;56(4):1376–86.10.1111/j.1471-4159.1991.tb11435.xSearch in Google Scholar PubMed

[55] Esfandiari A, Soifiyoudine D, Paturneau-Jouas M. Inhibition of fatty acid β-oxidation in rat brain cultured astrocytes exposed to the neurotoxin 3-nitropropionic acid. Dev Neurosci. 1997;19(4):312–20.10.1159/000111227Search in Google Scholar PubMed

[56] Eraso-Pichot A, Brasó-Vives M, Golbano A, Menacho C, Claro E, Galea E, et al. GSEA of mouse and human mitochondriomes reveals fatty acid oxidation in astrocytes. Glia. 2018;66(8):1724–35.10.1002/glia.23330Search in Google Scholar PubMed

[57] Knobloch M, Pilz G-A, Ghesquière B, Kovacs WJ, Wegleiter T, Moore DL, et al. A fatty acid oxidation-dependent metabolic shift regulates adult neural stem cell activity. Cell Rep. 2017;20(9):2144–55.10.1016/j.celrep.2017.08.029Search in Google Scholar PubMed PubMed Central

[58] Rumora AE, LoGrasso G, Hayes JM, Mendelson FE, Tabbey MA, Haidar JA, et al. The divergent roles of dietary saturated and monounsaturated fatty acids on nerve function in murine models of obesity. J Neurosci. 2019;39(19):3770–81.10.1523/JNEUROSCI.3173-18.2019Search in Google Scholar PubMed PubMed Central

[59] Sarangarajan R, Meera S, Rukkumani R, Sankar P, Anuradha G. Antioxidants: Friend or foe? Asian Pac J Trop Med. 2017;10(12):1111–6.10.1016/j.apjtm.2017.10.017Search in Google Scholar PubMed

[60] Lalkovičová M, Danielisová V. Neuroprotection and antioxidants. Neural Regener Res. 2016;11(6):865.10.4103/1673-5374.184447Search in Google Scholar PubMed PubMed Central

[61] Fatima N. Association of Peroxisomes, Reactive Oxygen Species (ROS) and antioxidants: Insights from preclinical and clinical evaluations. The metabolic role of peroxisome in health and disease. England: IntechOpen; 2022.10.5772/intechopen.105827Search in Google Scholar

[62] Dasari B, Prasanthi JR, Marwarha G, Singh BB, Ghribi O. The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells. BMC Ophthalmol. 2010;10(1):1–12.10.1186/1471-2415-10-22Search in Google Scholar PubMed PubMed Central

[63] Ferreiro E, Baldeiras I, Ferreira I, Costa R, Rego A, Pereira C, et al. Mitochondrial-and endoplasmic reticulum-associated oxidative stress in Alzheimer’s disease: from pathogenesis to biomarkers. Int J Cell Biol. 2012;2012:1–23.10.1155/2012/735206Search in Google Scholar PubMed PubMed Central

[64] Area-Gomez E, del Carmen Lara Castillo M, Tambini MD, Guardia-Laguarta C, De Groof AJ, Madra M, et al. Upregulated function of mitochondria-associated ER membranes in Alzheimer disease. EMBO J. 2012;31(21):4106–23.10.1038/emboj.2012.202Search in Google Scholar PubMed PubMed Central

[65] Liu X, Xu K, Yan M, Wang Y, Zheng X. Protective effects of galantamine against Aβ-induced PC12 cell apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum stress. Neurochem Int. 2010;57(5):588–99.10.1016/j.neuint.2010.07.007Search in Google Scholar PubMed

[66] Devore EE, Grodstein F, van Rooij FJ, Hofman A, Stampfer MJ, Witteman JC, et al. Dietary antioxidants and long-term risk of dementia. Arch Neurol. 2010;67(7):819–25.10.1001/archneurol.2010.144Search in Google Scholar PubMed PubMed Central

[67] Ikeda T, Yamamoto K, Takahashi K, Kaku Y, Uchiyama M, Sugiyama K, et al. Treatment of Alzheimer-type dementia with intravenous mecobalamin. Clin Therapeutics. 1992;14(3):426–37.Search in Google Scholar

[68] Picón-Pagès P, Garcia-Buendia J, Munoz FJ. Functions and dysfunctions of nitric oxide in brain. Biochim Biophys Acta-Mol Basis Dis. 2019;1865(8):1949–67.10.1016/j.bbadis.2018.11.007Search in Google Scholar PubMed

[69] Tajes M, Ill-Raga G, Palomer E, Ramos-Fernández E, Guix FX, Bosch-Morató M, et al. Nitro-oxidative stress after neuronal ischemia induces protein nitrotyrosination and cell death. Oxid Med Cell Longev. 2013;2013:1–9.10.1155/2013/826143Search in Google Scholar PubMed PubMed Central

[70] Frank MG. The mystery of sleep function: current perspectives and future directions. Rev Neurosci. 2006;17(4):375–92.10.1515/revneuro.2006.17.4.375Search in Google Scholar PubMed

[71] Zhang XW, Qiu QF, Jiang H, Zhang FL, Liu YL, Amatore C, et al. Real-time intracellular measurements of ROS and RNS in living cells with single core–shell nanowire electrodes. Angew Chem. 2017;129(42):13177–80.10.1002/ange.201707187Search in Google Scholar

[72] Khatri N, Thakur M, Pareek V, Kumar S, Sharma S, Datusalia AK. Oxidative stress: major threat in traumatic brain injury. CNS Neurol Disord-Drug Targets (Former Curr Drug Targets-CNS Neurol Disord). 2018;17(9):689–95.10.2174/1871527317666180627120501Search in Google Scholar PubMed

[73] Beal MF. Oxidatively modified proteins in aging and disease. Free Radic Biol Med. 2002;32(9):797–803.10.1016/S0891-5849(02)00780-3Search in Google Scholar PubMed

[74] Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet. 2006;38(5):515–7.10.1038/ng1769Search in Google Scholar PubMed

[75] González-Polo RA, Soler G, Rodrıguezmartın A, Morán JM, Fuentes JM. Protection against MPP + neurotoxicity in cerebellar granule cells by antioxidants. Cell Biol Int. 2004;28(5):373–80.10.1016/j.cellbi.2004.03.005Search in Google Scholar PubMed

[76] Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: A target for neuroprotection? Lancet Neurol. 2009;8(4):382–97.10.1016/S1474-4422(09)70062-6Search in Google Scholar PubMed

[77] Li HM, Niki T, Taira T, Iguchi-Ariga SM, Ariga H. Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress. Free Radic Res. 2005;39(10):1091–9.10.1080/10715760500260348Search in Google Scholar PubMed

[78] Lev N, Ickowicz D, Barhum Y, Melamed E, Offen D. DJ-1 changes in G93A-SOD1 transgenic mice: Implications for oxidative stress in ALS. J Mol Neurosci. 2009;38:94–102.10.1007/s12031-008-9138-7Search in Google Scholar PubMed

[79] Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, et al. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature. 2010;468(7324):696–700.10.1038/nature09536Search in Google Scholar PubMed PubMed Central

[80] Sharma C, Kim SR. Linking oxidative stress and proteinopathy in Alzheimer’s disease. Antioxidants. 2021;10(8):1231.10.3390/antiox10081231Search in Google Scholar PubMed PubMed Central

[81] Bhatt S, Puli L, Patil CR. Role of reactive oxygen species in the progression of Alzheimer’s disease. Drug Discov Today. 2021;26(3):794–803.10.1016/j.drudis.2020.12.004Search in Google Scholar PubMed

[82] Manoharan S, Guillemin GJ, Abiramasundari RS, Essa MM, Akbar M, Akbar MD. The role of reactive oxygen species in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease: a mini review. Oxid Med Cell Longev. 2016;2016:1–15.10.1155/2016/8590578Search in Google Scholar PubMed PubMed Central

[83] Xie Z, Wei M, Morgan TE, Fabrizio P, Han D, Finch CE, et al. Peroxynitrite mediates neurotoxicity of amyloid β-peptide1–42-and lipopolysaccharide-activated microglia. J Neurosci. 2002;22(9):3484–92.10.1523/JNEUROSCI.22-09-03484.2002Search in Google Scholar PubMed PubMed Central

[84] Van Dyke K. The possible role of peroxynitrite in Alzheimer’s disease: a simple hypothesis that could be tested more thoroughly. Med Hypotheses. 1997;48(5):375–80.10.1016/S0306-9877(97)90031-1Search in Google Scholar PubMed

[85] Ghanbari HA, Ghanbari K, Harris PL, Jones PK, Kubat Z, Castellani RJ, et al. Oxidative damage in cultured human olfactory neurons from Alzheimer’s disease patients. Aging Cell. 2004;3(1):41–4.10.1111/j.1474-9728.2004.00083.xSearch in Google Scholar PubMed

[86] Baldeiras I, Santana I, Proença MT, Garrucho MH, Pascoal R, Rodrigues A, et al. Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer’s disease. J Alzheimer’s Dis. 2008;15(1):117–28.10.3233/JAD-2008-15110Search in Google Scholar

[87] Migliore L, Fontana I, Trippi F, Colognato R, Coppede F, Tognoni G, et al. Oxidative DNA damage in peripheral leukocytes of mild cognitive impairment and AD patients. Neurobiol Aging. 2005;26(5):567–73.10.1016/j.neurobiolaging.2004.07.016Search in Google Scholar PubMed

[88] Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, et al. The Aβ peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry. 1999;38(24):7609–16.10.1021/bi990438fSearch in Google Scholar PubMed

[89] Opazo C, Huang X, Cherny RA, Moir RD, Roher AE, White AR, et al. Metalloenzyme-like activity of Alzheimer’s disease β-amyloid: Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H2O2. J Biol Chem. 2002;277(43):40302–8.10.1074/jbc.M206428200Search in Google Scholar PubMed

[90] Nelson TJ, Alkon DL. Oxidation of cholesterol by amyloid precursor protein and β-amyloid peptide. J Biol Chem. 2005;280(8):7377–87.10.1074/jbc.M409071200Search in Google Scholar PubMed

[91] Stamer K, Vogel R, Thies E, Mandelkow E, Mandelkow E-M. Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol. 2002;156(6):1051–63.10.1083/jcb.200108057Search in Google Scholar PubMed PubMed Central

[92] Petersen JD, Kaech S, Banker G. Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification. J Neurosci. 2014;34(12):4135–47.10.1523/JNEUROSCI.3779-13.2014Search in Google Scholar PubMed PubMed Central

[93] Kandimalla R, Manczak M, Yin X, Wang R, Reddy PH. Hippocampal phosphorylated tau induced cognitive decline, dendritic spine loss and mitochondrial abnormalities in a mouse model of Alzheimer’s disease. Hum Mol Genet. 2018;27(1):30–40.10.1093/hmg/ddx381Search in Google Scholar PubMed PubMed Central

[94] Melov S, Adlard PA, Morten K, Johnson F, Golden TR, Hinerfeld D, et al. Mitochondrial oxidative stress causes hyperphosphorylation of tau. PLoS one. 2007;2(6):e536.10.1371/journal.pone.0000536Search in Google Scholar PubMed PubMed Central

[95] David DC, Hauptmann S, Scherping I, Schuessel K, Keil U, Rizzu P, et al. Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice. J Biol Chem. 2005;280(25):23802–14.10.1074/jbc.M500356200Search in Google Scholar PubMed

[96] Klivenyi P, Clair DS, Wermer M, Yen H-C, Oberley T, Yang L, et al. Manganese superoxide dismutase overexpression attenuates MPTP toxicity. Neurobiol Dis. 1998;5(4):253–8.10.1006/nbdi.1998.0191Search in Google Scholar PubMed

[97] Callio J, Oury TD, Chu CT. Manganese superoxide dismutase protects against 6-hydroxydopamine injury in mouse brains. J Biol Chem. 2005;280(18):18536–42.10.1074/jbc.M413224200Search in Google Scholar PubMed PubMed Central

[98] Parihar VK, Allen BD, Tran KK, Chmielewski NN, Craver BM, Martirosian V, et al. Targeted overexpression of mitochondrial catalase prevents radiation-induced cognitive dysfunction. Antioxid Redox Signal. 2015;22(1):78–91.10.1089/ars.2014.5929Search in Google Scholar PubMed PubMed Central

[99] Przedborski S, Kostic V, Jackson-Lewis V, Naini AB, Simonetti S, Fahn S, et al. Transgenic mice with increased Cu/Zn-superoxide dismutase activity are resistant to N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced neurotoxicity. J Neurosci. 1992;12(5):1658–67.10.1523/JNEUROSCI.12-05-01658.1992Search in Google Scholar PubMed PubMed Central

[100] Liang L, Ho Y, Patel M. Mitochondrial superoxide production in kainate-induced hippocampal damage. Neuroscience. 2000;101(3):563–70.10.1016/S0306-4522(00)00397-3Search in Google Scholar PubMed

[101] Melov S, Doctrow SR, Schneider JA, Haberson J, Patel M, Coskun PE, et al. Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase–catalase mimetics. J Neurosci. 2001;21(21):8348–53.10.1523/JNEUROSCI.21-21-08348.2001Search in Google Scholar PubMed PubMed Central

[102] Wojtala A, Bonora M, Malinska D, Pinton P, Duszynski J, Wieckowski MR. Methods to monitor ROS production by fluorescence microscopy and fluorometry. Methods in enzymology. Netherlands: Elsevier; 2014. p. 243–62.10.1016/B978-0-12-416618-9.00013-3Search in Google Scholar PubMed

[103] Duanghathaipornsuk S, Farrell EJ, Alba-Rubio AC, Zelenay P, Kim D-S. Detection technologies for reactive oxygen species: fluorescence and electrochemical methods and their applications. Biosensors. 2021;11(2):30.10.3390/bios11020030Search in Google Scholar PubMed PubMed Central

[104] Rubio CP, Cerón JJ. Spectrophotometric assays for evaluation of Reactive Oxygen Species (ROS) in serum: General concepts and applications in dogs and humans. BMC Vet Res. 2021;17(1):226.10.1186/s12917-021-02924-8Search in Google Scholar PubMed PubMed Central

[105] Zhang Y, Dai M, Yuan Z. Methods for the detection of reactive oxygen species. Anal Methods. 2018;10(38):4625–38.10.1039/C8AY01339JSearch in Google Scholar

[106] Suzen S, Gurer-Orhan H, Saso L. Detection of reactive oxygen and nitrogen species by electron paramagnetic resonance (EPR) technique. Molecules. 2017;22(1):181.10.3390/molecules22010181Search in Google Scholar PubMed PubMed Central

[107] Lin W-Z, Yeung C-Y, Liang C-K, Huang Y-H, Liu C-C, Hou S-Y. A colorimetric sensor for the detection of hydrogen peroxide using DNA-modified gold nanoparticles. J Taiwan Inst Chem Eng. 2018;89:49–55.10.1016/j.jtice.2018.05.005Search in Google Scholar

[108] Huang J, Hou L, Bian X, Chang K. Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector. J Liq Chromatogr Relat Technol. 2019;42(13–14):429–35.10.1080/10826076.2019.1625369Search in Google Scholar

[109] Myhre O, Andersen JM, Aarnes H, Fonnum F. Evaluation of the probes 2′, 7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol. 2003;65(10):1575–82.10.1016/S0006-2952(03)00083-2Search in Google Scholar

[110] Iroegbu AOC, Ray SS. Recent developments and future perspectives of biorenewable nanocomposites for advanced applications. Nanotechnol Rev. 2022;11(1):1696–721.10.1515/ntrev-2022-0105Search in Google Scholar

[111] Guo H, Aleyasin H, Dickinson BC, Haskew-Layton RE, Ratan RR. Recent advances in hydrogen peroxide imaging for biological applications. Cell Biosci. 2014;4:1–10.10.1186/2045-3701-4-64Search in Google Scholar PubMed PubMed Central

[112] Niethammer P, Grabher C, Look AT, Mitchison TJ. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature. 2009;459(7249):996–9.10.1038/nature08119Search in Google Scholar PubMed PubMed Central

[113] Wu L, Sedgwick AC, Sun X, Bull SD, He X-P, James TD. Reaction-based fluorescent probes for the detection and imaging of reactive oxygen, nitrogen, and sulfur species. Acc Chem Res. 2019;52(9):2582–97.10.1021/acs.accounts.9b00302Search in Google Scholar PubMed PubMed Central

[114] Kim J-S, Jeong K, Murphy JM, Rodriguez YA, Lim S-TS. A quantitative method to measure low levels of ROS in nonphagocytic cells by using a chemiluminescent imaging system. Oxid Med Cell Longev. 2019;2019:1–18.10.1155/2019/1754593Search in Google Scholar PubMed PubMed Central

[115] Kumari R, Dkhar DS, Mahapatra S, Kumar R, Chandra P. Nano-bioengineered Sensing Technologies for Real-time Monitoring of Reactive Oxygen Species in vitro and in vivo models. Microchem J. 2022;180:107615.10.1016/j.microc.2022.107615Search in Google Scholar

[116] Geraskevich AV, Solomonenko AN, Dorozhko EV, Korotkova EI, Barek J. Electrochemical sensors for the detection of reactive oxygen species in biological systems: A critical review. Crit Rev Anal Chem. 2022;32:1–33.10.1080/10408347.2022.2098669Search in Google Scholar PubMed

[117] Naghib SM, Behzad F, Rahmanian M, Zare Y, Rhee KY. A highly sensitive biosensor based on methacrylated graphene oxide-grafted polyaniline for ascorbic acid determination. Nanotechnol Rev. 2020;9(1):760–7.10.1515/ntrev-2020-0061Search in Google Scholar

[118] Power AC, Gorey B, Chandra S, Chapman J. Carbon nanomaterials and their application to electrochemical sensors: a review. Nanotechnol Rev. 2018;7(1):19–41.10.1515/ntrev-2017-0160Search in Google Scholar

[119] Ramburrun P, Khan RA, Choonara YE. Design, preparation, and functionalization of nanobiomaterials for enhanced efficacy in current and future biomedical applications. Nanotechnol Rev. 2022;11(1):1802–26.10.1515/ntrev-2022-0106Search in Google Scholar

[120] Alhazmi HA, Ahsan W, Mangla B, Javed S, Hassan MZ, Asmari M, et al. Graphene-based biosensors for disease theranostics: Development, applications, and recent advancements. Nanotechnol Rev. 2021;13(6):96–116.10.1515/ntrev-2022-0009Search in Google Scholar

[121] Madani M, Hosny S, Alshangiti DM, Nady N, Alkhursani SA, Alkhaldi H, et al. Green synthesis of nanoparticles for varied applications: Green renewable resources and energy-efficient synthetic routes. Nanotechnol Rev. 2022;4:731–59.10.1515/ntrev-2022-0034Search in Google Scholar

[122] Malferrari M, Becconi M, Rapino S. Electrochemical monitoring of reactive oxygen/nitrogen species and redox balance in living cells. Anal Bioanal Chem. 2019;411:4365–74.10.1007/s00216-019-01734-0Search in Google Scholar PubMed

[123] Bedioui F, Quinton D, Griveau S, Nyokong T. Designing molecular materials and strategies for the electrochemical detection of nitric oxide, superoxide and peroxynitrite in biological systems. Phys Chem Chem Phys. 2010;12(34):9976–88.10.1039/c0cp00271bSearch in Google Scholar PubMed

[124] Liu X, Dumitrescu E, Andreescu S. Electrochemical biosensors for real-time monitoring of reactive oxygen and nitrogen species. Oxidative Stress: Diagnostics, Prevention, and Therapy. Vol. 2. USA: ACS Publications; 2015. p. 301–27.10.1021/bk-2015-1200.ch013Search in Google Scholar

[125] Rhee SG. Redox signaling: Hydrogen peroxide as intracellular messenger. Exp Mol Med. 1999;31(2):53–9.10.1038/emm.1999.9Search in Google Scholar PubMed

[126] Góth L. The hydrogen peroxide paradox. Orvosi Hetil. 2006;147(19):887–93.Search in Google Scholar

[127] Veal E, Day A. Hydrogen peroxide as a signaling molecule. Antioxid Redox Signal. 2011;15(1):147–51.10.1089/ars.2011.3968Search in Google Scholar PubMed

[128] Buetler TM, Krauskopf A, Ruegg UT. Role of superoxide as a signaling molecule. Physiology. 2004;19(3):120–3.10.1152/nips.01514.2003Search in Google Scholar PubMed

[129] Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 2017;11:613–9.10.1016/j.redox.2016.12.035Search in Google Scholar PubMed PubMed Central

[130] Chen BT, Avshalumov MV, Rice ME. H2O2 is a novel, endogenous modulator of synaptic dopamine release. J Neurophysiol. 2001;85(6):2468–76.10.1152/jn.2001.85.6.2468Search in Google Scholar PubMed

[131] Patel JC, Rice ME. Classification of H2O2 as a neuromodulator that regulates striatal dopamine release on a subsecond time scale. ACS Chem Neurosci. 2012;3(12):991–1001.10.1021/cn300130bSearch in Google Scholar PubMed PubMed Central

[132] O’Riordan SL, Lowry JP. In vivo characterisation of a catalase-based biosensor for real-time electrochemical monitoring of brain hydrogen peroxide in freely-moving animals. Anal Methods. 2017;9(8):1253–64.10.1039/C6AY03066ASearch in Google Scholar

[133] Li R, Liu X, Qiu W, Zhang M. In vivo monitoring of H2O2 with polydopamine and prussian blue-coated microelectrode. Anal Chem. 2016;88(15):7769–76.10.1021/acs.analchem.6b01765Search in Google Scholar PubMed

[134] Zhang S, Tao-Tao F, Zhang L, Zhang M-N. In vivo electrochemical detection of hydrogen peroxide and dopamine. Chin J Anal Chem. 2019;47(10):1664–70.10.1016/S1872-2040(19)61193-XSearch in Google Scholar

[135] Li S, Tian Y. An electrochemical biosensor with dual signal outputs for ratiometric monitoring the levels of H2O2 and pH in the microdialysates from a rat brain. Electroanalysis. 2018;30(6):1047–53.10.1002/elan.201700684Search in Google Scholar

[136] Wilson LR, Panda S, Schmidt AC, Sombers LA. Selective and mechanically robust sensors for electrochemical measurements of real-time hydrogen peroxide dynamics in vivo. Anal Chem. 2018;90(1):888–95.10.1021/acs.analchem.7b03770Search in Google Scholar PubMed PubMed Central

[137] Luo Y, Lin R, Zuo Y, Zhang Z, Zhuo Y, Lu M, et al. Efficient electrochemical microsensor for In vivo monitoring of H2O2 in PD mouse brain: Rational design and synthesis of recognition molecules. Anal Chem. 2022;94(25):9130–9.10.1021/acs.analchem.2c01570Search in Google Scholar PubMed

[138] Li X, Zhou L, Ding J, Sun L, Su B. Platinized silica nanoporous membrane electrodes for low-fouling hydrogen peroxide detection. ChemElectroChem. 2020;7(9):2081–6.10.1002/celc.202000321Search in Google Scholar

[139] Rahman MA, Kothalam A, Choe ES, Won M-S, Shim Y-B. Stability and sensitivity enhanced electrochemical in vivo superoxide microbiosensor based on covalently co-immobilized lipid and cytochrome c. Anal Chem. 2012;84(15):6654–60.10.1021/ac301086mSearch in Google Scholar PubMed

[140] Peng Q, Yan X, Shi X, Ou S, Gu H, Yin X, et al. In vivo monitoring of superoxide anion from Alzheimer’s rat brains with functionalized ionic liquid polymer decorated microsensor. Biosens Bioelectron. 2019;144:111665.10.1016/j.bios.2019.111665Search in Google Scholar PubMed

[141] Huang S, Zhang L, Dai L, Wang Y, Tian Y. Nonenzymatic electrochemical sensor with ratiometric signal output for selective determination of superoxide anion in rat brain. Anal Chem. 2021;93(13):5570–6.10.1021/acs.analchem.1c00151Search in Google Scholar PubMed

[142] Brown MD, Schoenfisch MH. Electrochemical nitric oxide sensors: principles of design and characterization. Chem Rev. 2019;119(22):11551–75.10.1021/acs.chemrev.8b00797Search in Google Scholar PubMed

[143] Meiller A, Sequeira E, Marinesco S. Electrochemical nitric oxide microsensors based on a fluorinated xerogel screening layer for in vivo brain monitoring. Anal Chem. 2019;92(2):1804–10.10.1021/acs.analchem.9b03621Search in Google Scholar PubMed

[144] Ha Y, Sim J, Lee Y, Suh M. Insertable fast-response amperometric NO/CO dual microsensor: Study of neurovascular coupling during acutely induced seizures of rat brain cortex. Anal Chem. 2016;88(5):2563–9.10.1021/acs.analchem.5b04288Search in Google Scholar PubMed

[145] Ha Y, Lee Y, Suh M. Insertable NO/CO microsensors recording gaseous vasomodulators reflecting differential neuronal activation level with respect to seizure focus. ACS Chem Neurosci. 2017;8(9):1853–8.10.1021/acschemneuro.7b00141Search in Google Scholar PubMed

[146] Moon J, Ha Y, Kim M, Sim J, Lee Y, Suh M. Dual electrochemical microsensor for real-time simultaneous monitoring of nitric oxide and potassium ion changes in a rat brain during spontaneous neocortical epileptic seizure. Anal Chem. 2016;88(18):8942–8.10.1021/acs.analchem.6b02396Search in Google Scholar PubMed

[147] Park SS, Hong M, Song C-K, Jhon G-J, Lee Y, Suh M. Real-time in vivo simultaneous measurements of nitric oxide and oxygen using an amperometric dual microsensor. Anal Chem. 2010;82(18):7618–24.10.1021/ac1013496Search in Google Scholar PubMed

[148] Jo A, Do H, Jhon G-J, Suh M, Lee Y. Electrochemical nanosensor for real-time direct imaging of nitric oxide in living brain. Anal Chem. 2011;83(21):8314–9.10.1021/ac202225nSearch in Google Scholar PubMed

[149] Santos RM, Rodrigues MS, Laranjinha J, Barbosa RM. Biomimetic sensor based on hemin/carbon nanotubes/chitosan modified microelectrode for nitric oxide measurement in the brain. Biosens Bioelectron. 2013;44:152–9.10.1016/j.bios.2013.01.015Search in Google Scholar PubMed

[150] Lamanna G, Battigelli A, Ménard-Moyon C, Bianco A. Multifunctionalized carbon nanotubes as advanced multimodal nanomaterials for biomedical applications. Nanotechnol Rev. 2012;1(1):17–29.10.1515/ntrev-2011-0002Search in Google Scholar

[151] Amali R, Lim H, Ibrahim I, Huang N, Zainal Z, Ahmad S. Significance of nanomaterials in electrochemical sensors for nitrate detection: A review. Trends Environ Anal Chem. 2021;31:e00135.10.1016/j.teac.2021.e00135Search in Google Scholar

[152] Pandey RR, Chusuei CC. Carbon nanotubes, graphene, and carbon dots as electrochemical biosensing composites. Molecules. 2021;26(21):6674.10.3390/molecules26216674Search in Google Scholar PubMed PubMed Central

[153] Huang S, Du X, Ma M, Xiong L. Recent progress in the synthesis and applications of vertically aligned carbon nanotube materials. Nanotechnol Rev. 2021;10(1):1592–623.10.1515/ntrev-2021-0102Search in Google Scholar

[154] Li R, Qi H, Ma Y, Deng Y, Liu S, Jie Y, et al. A flexible and physically transient electrochemical sensor for real-time wireless nitric oxide monitoring. Nat Commun. 2020;11(1):3207.10.1038/s41467-020-17008-8Search in Google Scholar PubMed PubMed Central

[155] Liu F, Dong H, Tian Y. Real-time monitoring of peroxynitrite (ONOO−) in the rat brain by developing a ratiometric electrochemical biosensor. Analyst. 2019;144(6):2150–7.10.1039/C9AN00079HSearch in Google Scholar

[156] Li Y, Hu K, Yu Y, Rotenberg SA, Amatore C, Mirkin MV. Direct electrochemical measurements of reactive oxygen and nitrogen species in nontransformed and metastatic human breast cells. J Am Chem Soc. 2017;139(37):13055–62.10.1021/jacs.7b06476Search in Google Scholar PubMed

[157] Vaneev AN, Gorelkin PV, Garanina AS, Lopatukhina HV, Vodopyanov SS, Alova AV, et al. In vitro and in vivo electrochemical measurement of reactive oxygen species after treatment with anticancer drugs. Anal Chem. 2020;92(12):8010–4.10.1021/acs.analchem.0c01256Search in Google Scholar PubMed

[158] Reiniers MJ, van Golen RF, van Gulik TM, Heger M. Reactive oxygen and nitrogen species in steatotic hepatocytes: a molecular perspective on the pathophysiology of ischemia-reperfusion injury in the fatty liver. Antioxid Redox Signal. 2014;21(7):1119–42.10.1089/ars.2013.5486Search in Google Scholar PubMed PubMed Central

Received: 2022-12-13

Revised: 2023-07-08

Accepted: 2023-08-26

Published Online: 2023-10-25

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/ntrev-2023-0124

Keywords for this article

electrochemical; biosensors; oxygen species

Creative Commons

BY 4.0

Electrochemical micro- and nanobiosensors for in vivo reactive oxygen/nitrogen species measurement in the brain

Article

Abstract

1 Introduction

2 Formation mechanisms of ROS and RNS

3 The importance of ROS and RNS in the brain

4 ROS/RNS detection methods

5 Electrochemical measurement of ROS in the brain

5.1 Electrochemical measurement of H2O2 in the brain

5.2 Electrochemical measurement of superoxide

5.3 Comparative evaluation of electrochemical sensor designs for ROS detection in the brain

6 Electrochemical measurement of RNS in the brain

6.1 Electrochemical measurement of nitric oxide in the brain

6.2 Electrochemical measurement of peroxynitrite in the brain

7 Outlook and conclusion

References

Articles in the same Issue

Articles in the same Issue

Articles in the same Issue

5.1 Electrochemical measurement of H₂O₂ in the brain