Functional nitrogen science based on plasma processing: quantum devices, photocatalysts and activation of plant defense and immune systems

The Haber–Bosch (HB) process, initially developed in the 1910s, is based on the reaction N₂ + 3H₂ → 2NH₃ at pressures above 10 MPa and temperatures between 400 °C and 500 °C with an iron-based catalyst bed. This has since become a common nitrogen fixation method but has the drawback of requiring extreme conditions. Thus, the synthesis of ammonia at ambient temperature and atmospheric pressure would be highly beneficial and has been widely researched. Bacterial nitrogenase enzymes are known to have a Fe–Mo complex center, as shown in Fig. 3(a) and the conversion of dinitrogen to ammonia occurs through a catalytic cycle involving reactive intermediates, such as diazenido (–N=NH), hydrazido (–N–NH₂), hydrazidium (–N–NH₃), nitrido (≡N), imido (=NH), amido (–NH₂), and ammonium (–NH₃) complexes. ^3,4) The Nishibayashi group recently demonstrated a molybdenum-based catalyst that promotes the formation of ammonia from dinitrogen at temperatures and pressures close to ambient. ⁵⁾ In addition, Legare et al. reported that a solid phase reductant and acid reagents such as B(OH)₃ can be used as protonating agents in the reduction of dinitrogen to ammonia. Legare et al. demonstrated a complex series of reactions involving protonation to form hydraziono diradicals in a one-pot synthesis. ⁶⁾

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In plants, rhizobium bacteria act to fix nitrogen to generate nitrite nitrogen (NO₂–N) or nitrate nitrogen (NO₃–N), both of which are essential nutrients. However, the direct use of dinitrogen as a raw material for fertilizer has not yet been achieved. The enzymatic reactions involving nitrogenases by which rhizobium bacteria fix nitrogen are believed to be catalyzed by iron-sulfur clusters or molybdenum metal complexes. ^7–9) (Fig. 3) In this process, an iron site binds with dinitrogen and catalyzes the formation of ammonia via the generation of a hydraziono diradical and distal nitride (Fe≡N + NH₃). (Fig. 3)

Prior to the invention of the HB process, electrical discharges that generated plasmas were used as a means of nitrogen fixation. ¹⁰⁾ After the HB process was introduced, the electrical discharge-based nitrogen fixation became obsolete, since this technique could not be efficiently scaled up and also required a significant amount of electrical energy. ^11,12) In 1900s, gaseous electrical discharges were limited by the use of simple direct current sources to control electrical currents. Discharge technologies developed over time, with the use of short-pulsed high-voltage, high-frequency, and microwave discharge power sources. At present, the energy produced by plasma generation has become efficient. However, the economic cost of the plasma-based process is higher than that of the HB process, which is directly powered by fossil fuels. An electrical discharge-based process is required to use costly electricity; an indirect use of primary energy sources. From another viewpoint, the plasma-based process can be used at any location where nitrogen is necessary. Therefore, this review discusses the in-situ utilization of nitrogen using atmospheric air plasma technology.

Briefly, the electric breakdown of gases results in an avalanche of ionization due to accelerating primary electrons in a static electrical force caused by applying high electric voltages. ¹³⁾ Figure 4 shows the schematics of the plasma sources. The ionized charges cause coronal plasma and transient sparks, forming filamentary discharges. An electrode is at least covered by a low-electrical-conductivity material. Electric current flows only when the polarity of the applied voltage changes and a plasma discharge is ignited. This basic mechanism is called the dielectric barrier discharge (DBD). In addition, plasma is generated independently and remotely from the target, and reactive species transfer indirectly to the target surface through effluent gases. Moreover, there is another configuration in which processed droplets flow inside the plasma region and the processed droplet or mist transfers to the surface. At atmospheric pressure, the electric breakdown is not stabilized, and a transition of the thermal arc occurs as the discharge current increases. Therefore, the prevention of thermalization, which is caused by the higher electron collision-induced momentum transfer than the lower cooling mechanisms on the ambient and surrounding walls outside the discharge region, is extremely important. Meanwhile, electron-related phenomena, including ionization, dissociation, and excitation, produce reactive species at lower temperatures than thermal processes. In atmospheric air, plasmas, reactive oxygen, and nitrogen species (Table I) are produced. ¹⁴⁾ A detailed list has been provided by Ishikawa. ¹⁵⁾

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Even so, plasma discharge may be a viable approach to enhancing catalytic reactions at low temperatures because such discharges can promote the dissociation of the stable N≡N bond (bond dissociation energy of 9.76 eV) and also generate ultraviolet radiation. As such, the concept of plasma-assisted nitrogen fixation at low or moderate temperatures and atmospheric pressure has recently attracted much attention, especially with regard to plasma catalysis and plasma-treated solutions.

Plasma discharges have been applied to chemical reactions in several contexts. Plasma can dissociate molecules by providing bond-breaking energy through stepwise vibrational excitation. This leads to (1) the formation of pre-dissociative species in the gaseous phase and (2) radical-enhanced reactions. The surface of the catalysts is exposed by electrons, ions, and the chemically activated neutral species generated in the plasma. Impingement activates the surface, leading to (3) activation of the catalytic surface induced by thermal energy, (4) photocatalytic activation, (5) build-up of electric field and electronic charges on the surface to assist in reduction and (6) acid-base pair formation. The effective catalytic formation of ammonia can be regarded as acid hydrolysis of the intermediate complexes. (Fig. 3)

As an example, Kim et al. reported that a ruthenium-based catalyst could be used for ammonia synthesis in conjunction with a plasma-assisted catalytic reactor. ¹⁶⁾ The Haruyama group also demonstrated ammonia production using ultraviolet radiation at the interface between a plasma and an aqueous phase. ¹⁷⁾ In 2015, Patil et al. published a review of plasma-assisted nitrogen fixation ¹⁸⁾ and further extensive reviews of nanocatalysts for plasma-assisted nitrogen fixation were published in 2020. ^19,20) Hong et al. demonstrated that the electric field associated with a plasma may enhance catalytic surface reactivity by accelerating the donation and acceptance of electrons. ^21–23) Zhang and Oehrlein reviewed plasma-assisted catalytic surface reactions and compared these with thermal processes, ²⁴⁾ and another review provided a summary of progress in the study of plasma-assisted catalytic reactions. ^25–27) Kambara et al. reported the plasma-assisted decomposition of ammonia for the production of hydrogen using a plasma membrane reactor. ²⁸⁾ Plasma catalysis is gaining interest as a sustainable method for material synthesis with lower energy input. However, research on the topic has not yet assessed any industrialization issues, such as catalyst life and economic cost. The advantage of plasma catalysis is that the catalytic surface is effectively activated by facilitating chemically reactive radicals and other energetic particle irradiations, leading to activation of the catalytic reaction on the surface via the enhancement of electron transfer from sources to products.

Plasma treatment of water under ambient air has attracted attention, based on the use of atmospheric air plasma sources. Ikawa et al. reported that plasma-activated water (PTW) is able to kill bacteria at low pH values due to the formation of hydroperoxy (HOO·) radicals. ²⁹⁾ In addition, Lukes et al. established that plasma-activated water (PAW) contained H₂O₂ and HNO₂ in addition to peroxynitrite (ONOO⁻) ions. ³⁰⁾ Kurake et al. demonstrated that applying a plasma to cell culture media generated both H₂O₂ and HNO₂, which killed cancer cells through a synergistic process. ³¹⁾ The reaction mechanisms of these species in the gaseous and aqueous phases were also examined ³²⁾ and it was determined that reactive oxygen species (ROS) were able to selectively kill cancer cells in the presence of normal cells. The synergistic effects of ROS and RNS together with other organic compounds play an essential role in this selective phenomenon, suggesting that plasma-based cancer therapies may be viable. Tables II and III tabulate aqueous concentrations and primary routes of representative ROS and RNS. Bradu et al. reviewed the use of PTW in agriculture. ³³⁾ RNS have been found to activate various physiological processes in plants, such as seed germination and plant growth. Progress in the study of the PTW and PAW has been reviewed elsewhere. ^34,35)

Table II. List of aqueous concentrations of representative reactive oxygen and nitrogen species (RONS) in the plasma-activated water (PTW) and PAW. DBD stands for dielectric barrier discharge.

References	Machala 36)	Lukes 37)	Chauvin 38)	Heirman 39)	Kruszelnicki 40)
Plasma source	Spark	Corona	DBD	Effluent (kINPen)	Droplet
H2O2	600 μM	0.22 mM	1.6 mM	600 nM	570 mM
O2 −	—	—	—	40 nM	1.1 μM
OH	—	—	—	—	0.3 μM
NO2 −	300 μM	0.10 mM	0.5 mM	50 nM	—
NO3 −	1.2 mM	0.14 mM	0.4 mM	20 nM	570 mM
ONOO−	—	—	—	1 nM	0.09 μM

Table III. Primary route for loss of a part of ROS and RNS. ⁴¹⁾

Species	Primary route for loss	Lifetime
H2O2	2H2O2 → O2 + 2H2O	Stable
O2 .−	H+ + O2 − ↔ HO2	10–6 s
	NO + O2 − → ONOO−
OH	OH + OH → H2O2	10–10 s
NO2 −	H+ + NO2 − ↔ HNO2	Stable
	NO + NO2 − → N2O + O2 –
	2 NO2 − → N2 + 2 O2 –
NO3 −	H+ + NO3 − ↔ HNO3	Stable
ONOO−	H+ + ONOO− ↔ ONOOH	10–3 s

Electron collisions are able to rupture the stable triple bond of nitrogen under some conditions. This process involves excitation, dissociation, ionization and recombination and is capable of synthesizing a variety of RNS together with metastable forms of excited state nitrogen and vibrationally excited nitrogen molecules.

Recent progress in the plasma generation of RNS has shown that a combination of two plasma sources in conjunction with humid conditions can efficiently generate N₂O₅. ⁴²⁾ Takashima et al. reported that spraying water droplets into an atmospheric air plasma suppressed the formation of ozone ⁴³⁾ and other groups have developed different plasma sources on this basis. ^44–46) The low-temperature-operated source generates a high level of ozone while the other high-temperature-operated source can provide high NO concentrations. A plasma that is rich in N₂O₅ can also be used as an RNS source because the dissolution of N₂O₅ in water immediately generates nitrite and nitrate anions (NO₂ ⁻, and NO₃ ⁻). This PTW can be used for the sterilization of bacteria and inactivation of viruses, as well as the activation of plant immune systems. ⁴⁷⁾ Furthermore, although a different plasma source was used, another study demonstrated that NH₄NO₃ was generated in plasma-treated ammonia water. ⁴⁸⁾ There have been reports of the formation of a wide variety of RNS using atmospheric air plasma discharges.

Presently, various types of plasma sources have been developed by many researchers and engineers. Among the requirements for this development, one is the selective formation of selected species, as described above in the case of N₂O₅. Uchida et al. reported that the stoichiometry of N₂ and O₂ changed the concentration ratio of NO₂ ⁻ to H₂O₂. ^49–51) However, a comprehensive understanding of this process and sophisticated control of RNS generation have not yet been achieved. In such systems, the RNS that reach the reaction surface adhere to or penetrate into the substrate, and this process is termed nitrogen doping. This doping, in turn, can lead to functionalization that modifies properties such as hardness, electrical resistance and magnetism. Considering this situation, the directionality of plasma source development should be to precisely control any quantitative dosages for each reactive species at any spatial location in material applications. As described in Sect. 3.3, nitrogen-doped in a lattice system is sensitive to the location of nitrogen atoms, for example, whether substitutional or interstitial sites modify the photocatalytic activity of TiO₂. Further developments are required to improve the atomic control of nitrogen localization in lattice systems. In biological applications, the homeostatic responses of living organisms play a key role in the dosing of reactive species generated in plasma. Even as the homeostatic regulation of living organisms is retained under natural environmental stresses, mixed concentrations of ingredients or constituents should be dosed. As described in Sect. 6.1, RNS in the plant defense system and phytohormone balance for seed germination are seen in natural responses. At the molecular level, artificial RNS also affects these regulations in plant and cell systems. The challenge arises from the complexity of biological responses that are represented by reaction networks, as described in Sect. 5. In other words, macroscopic physiology as phenotypes in plant immune responses and seed germination promotion may control the mesoscopic reconnection of biochemical reaction networks through microscopic modifications induced by molecular level reactions. Thus, novel methods that combine measurements and manipulations are required for plasma sources.

Quantum information technology has become a hot topic. Quantum bits (qubits) are based on the superposition of multiple states, which is equivalent to the quantum entanglement associated with the Einstein, Podolsky and Rosen paradox. ⁵²⁾ In quantum mechanics, quantum states can be represented by the superposition of basis vectors. As an example, if the vectors ∣0> and ∣1> are defined, these vectors can undergo coherent superposition to produce a qubit. Qubits have been realized by controlling the spin of electronic charges, based on employing superconductor junctions, ion-traps, photons or quantum dots. ⁵³⁾

Fullerenes are a family of mesh-like carbon allotropes, and endohedral fullerenes can be obtained by incorporating nitrogen atoms into C₆₀, to isolate the electronic spin of single nitrogen atoms. ⁵⁴⁾ Coupling between this endohedral fullerene (N@C₆₀) of spin S = 3/2 and C₆₀ ⁻ radical anion (S = 1/2), for instance, offers implementation of quantum spin manipulation. ^55,56) Since multiple quantum states can be superposed, this configuration can act as a qubit with a long quantum decoherence time. It has been reported that nitrogen can be incorporated into fullerenes using plasma technology, ^57–60) although it should be noted that this process is different from technologies that incorporate nitrogen into various other molecules. The isolated spin of a nitrogen atom and the nuclear spins of isotopes such as ³¹P, ²⁹Si and ¹³C in a suitable matrix can be used as qubits, as described in the section below.

Recently, diamond has attracted attention as a potential component of quantum computing devices ^61–64) because dangling bonds and nitrogen complex centers in this substance can provide extremely long coherence times. These NV centers can have negative, neutral or positive charges, and negatively charged NV centers are the most effective at storing quantum information. Figure 5 shows a schematic illustration of the neutral NV center (NV⁰) and negatively charged NV center (NV⁻). For NV⁰, transitions between the triple electronic ground state ³A₂ and the excited state ³E (λ₁ ∼ 637 nm), and the singlet ¹E and ¹A₁ (λ₂ ∼ 1064 nm), are optically active. Applying the external magnetic field of B, Zeeman shifts are observed by the degeneracy of the spin momentum sublevels, m_s = ±1. Furthermore, as an electron is trapped at the vacancy site, coupling between the nuclear spin I of nitrogen and an electron spin is split into sublevels of the degeneracy of spin quantum numbers, m_s and m_I. ⁶⁵⁾ This two-spin system holds quantum states and the optically active transitions (thick arrows) can be used for both the operation and observation of qubits.

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Neumann et al. reported that the optical detection of magnetic resonance analysis of NV centers is sensitive to temperature and external magnetic and electric fields. ⁶¹⁾ Using the environmental sensitivity of the NV centers in diamond, nano-quantum sensors can be realized and this phenomenon has been widely researched, especially with regard to quantum magnetometry. ⁶¹⁾ The prolonged coherence of two qubits can also be used for the secure transmission of data, and the formation of EPR pairs from multiple qubits is referred to as a quantum network. The information on such networks can be stored and transmitted securely.

3.1.1. Formation of nitrogen-vacancy center in artificial diamond

3.1.2. Plasma CVD engineering of diamond

The doping of impurities into a crystal lattice can change the characteristics of a material. Such effects are based on adjusting the stoichiometry of the substance being processed and can tune the acoustic, elemental, chemical, electrical (such as resistance, piezo and pyro characteristics), magnetic, mechanical, optical and thermal properties of the material.

Materials such as semiconductors are vital to the fabrication of ultra-small, ultra-high speed, ultra-low power electronic devices, and these may also require nitrogen doping. Nitrogen doping in a silicon dioxide film used as a gate insulator can effectively suppress the diffusion of boron originating from a doped poly-silicon gate through the insulator film. In the case of ultra-large-scale integrated semiconductor devices, nitrogen-doped silicon dioxide films serve as gate-insulating materials. ^93–95) These films are referred to as silicon oxynitride (SiON) layers. The concentration profile of nitrogen in the film is localized at the interface between the metal or semiconductor and the bulk insulating SiON film, as exampled by a stack of 0.5 nm-level SiN layer and bulk 1.0 nm-level bulk SiON. Optimization of this profile can allow the realization of low power and high drivability transistors. In future devices, nitrogen doping will need to be controlled at the sub-nanometer scale.

Nitrogen-doped TiO₂ acting as a visible light-responsive photocatalyst has been prepared via N⁺ implantation and found to promote the photocatalytic degradation of methylene blue (MB) under visible light. X-ray absorption near-edge structure (XANES) analyses indicated that the nitrogen in this material had two possible chemical states: photocatalytically active N substituted at active O sites and inactive NO₂ and/or N–N species. Investigations using the depth-resolved N K-edge energy loss near edge structure (ELNES) technique confirmed these two types of N, which varied depending on the N concentration, and the local N concentration that provided an effective visible light response was estimated to be less than 1.8 atom%.

3.3.1. Nitrogen ion-implanted TiO₂ photocatalysts

3.3.2. Nitrogen-implanted TiO₂

The samples were comprised of TiO₂(100) single crystals (5 × 5 × 0.5 mm³). Mass analyzed 100 keV N₂ ⁺ ions (50 keV/N⁺ ion⁻¹) were injected into the samples at room temperature, perpendicular to the sample surface at an N⁺ fluence ranging from 1 to 5 × 10²¹ m⁻². After ion implantation, some samples were heat-treated at 573 K for 2 h in air. Optical absorption spectra of the samples before and after N⁺ implantation were acquired at room temperature using a spectrometer (JASCO V-670). A typical photocatalytic reaction experiment consisted of placing an N⁺-implanted sample in 0.5 ml of an aqueous MB solution (9.8 μ mol l⁻¹) followed by exposure to visible light generated using a 15 W Xe lamp with a cut filter for λ > 430 nm. Figure 6(a) presents the optical absorption spectra of the TiO₂ samples before and after the N⁺ implantation process. The absorption edge corresponding to the bandgap of TiO₂ was observed at approximately 410 nm prior to implantation and a new absorption band in the visible light region became more intense as the nitrogen dose was increased. The photocatalytic activity of this material was maximized at a fluence of 3 × 10²¹ m⁻² and then decreased with further increases in the fluence, suggesting that there was an optimum nitrogen concentration [Fig. 6(b)]. The sample doped using a fluence of 5 × 10²¹ m⁻² followed by heat treatment at 573 K was almost photocatalytically inactive under visible light. This confirmed that visible light responsiveness was not directly connected to the photo-absorbance of the material.

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3.3.3. Active and inactive nitrogen sites in TiO₂

N K-edge XANES spectra of the N⁺-implanted TiO₂ samples were obtained on the UVSOR-II BL-8B1 station at the Institute for Molecular Science and on the BL-7U station of the Aichi Synchrotron Radiation Center. Data were recorded at room temperature in the total electron yield mode to probe near the surface of the samples. ¹⁰⁸⁾ Figure 7 provides N K-edge XANES spectra of the N⁺-implanted TiO₂ samples and of TiN powder. The similar features of these spectra suggest that the N in the sample treated using a fluence of 3 × 10²¹ m⁻² (the active photocatalyst) was in a chemical environment similar to that in TiN. More thorough observations showed that a double peak at approximately 400 eV generated by the sample was shifted to lower energy compared with that for TiN. In contrast, the XANES spectrum of the sample treated with an N⁺ fluence of 5 × 10²¹ m⁻² followed by heat treatment (the inactive photocatalyst) showed a distinct single peak at approximately 401 eV. Therefore, a double-peak structure around 400 eV corresponds to an active nitrogen species while a single peak at 401 eV represents an inactive nitrogen species. These spectral features were well reproduced by theoretical calculations using the FEFF code. ¹⁰⁹⁾ The double peak obtained from the active photocatalyst was reproduced using a model in which one O atom in TiO₂ was substituted with an N, while the single peak at 401 eV generated by the inactive photocatalyst was attributed to the formation of NO₂ and/or N–N bonds. These results indicated that the occupation of the oxygen sites by nitrogen effectively provided visible light responsiveness.

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The optimum nitrogen concentration required to generate photocatalytically active nitrogen was investigated via quantitative analysis of the depth-resolved ELNES spectra of the active photocatalyst. Depth-resolved EEL spectra of sample cross-sections were recorded using a Gatan ENFINA 1000 spectrometer attached to a JEM200CX transmission electron microscope operated at 200 kV. ¹¹⁰⁾ The local N concentrations were also evaluated, as shown in Fig. 8(a). A Monte Carlo calculation (SRIM code ¹¹¹⁾) indicated that the implanted N atoms were distributed to depths of 180 nm from the surface, with the peak concentration at approximately 90 nm. Figure 8(b) presents the depth-resolved profiles extracted from the N K-edge ELNES data for the active photocatalyst. A double-peak structure around 398–401 eV was again observed near the surface region, in good agreement with the XANES spectrum of the same sample. A distinct single peak at approximately 401 eV gradually increased in intensity with increasing depth, while a single peak feature was observed during the XANES analysis of the photocatalytically inactive sample. These results suggested that the two different spectral profiles corresponded to variations in the local nitrogen concentrations as well as the photo-catalytic responsiveness. The single peak at 401 eV was predominant at depths below approximately 25 nm, and so the local concentration of doped nitrogen that most effectively provided a visible light response was estimated to be less than 1.8 atom%.

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3.3.4. Atomic-scale localization of nitrogen for effective photocatalysis

The preceding sections reviewed nitrogen doping into quantum devices and photocatalysts, in which the atomic position of nitrogen in the matrix lattice plays a significant role. Generally, metal oxides have a band gap between the valence and conduction bands. When an oxygen atom is eventually removed from the lattice position, an oxygen vacancy is formed (as schematically shown in Fig. 9), and unoccupied dangling orbitals create deep defect states in the band gap. By substituting the oxygen atom of the lattice with a nitrogen atom, the dangling bond of nitrogen generates a mid-gap energy state. In addition, interstitial nitrogen can be incorporated into the lattice and eventually, oxygen vacancies can be formed. For transition metals, atomic configurations become complex, owing to the d-orbitals. However, for simplicity, once electrons migrate from the valence band to the conduction band, for example, optical absorption, electron-hole (e⁻– h⁺) pairs are generated. In catalytic reactions, these electrons yield highly oxidative species. For example, redox reactions involving the reduction of O₂ + e⁻ to O₂ ⁻ and oxidation of OH⁻ + h⁺ to OH can occur, based on nitrogen dangling bonds and electrons in the conduction band. Thus, the localization of doping nitrogen into the lattice is essential to obtain chemical activity.

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In addition to ammonia synthesis, the photosynthesis of carbohydrates from CO₂ can occur, during which porphyrins (made of four pyrroles interconnected by methine bonds) are key molecular units (Fig. 10). In a porphyrin, the nitrogen is coordinated with a central metal atom, and these compounds undergo reactions with either ammonia or CO₂ involving electron transfer. ¹¹²⁾ Chlorophyll is a photosystem reaction center and nitrite reductase is a type of porphyrin that mediates electron transfer from iron-sulfur metalloprotein (4Fe-4S cluster center enzyme). Although porphyrins are easily oxidized or reduced, they are stable when generating the corresponding radical cations or radical anions. ¹¹³⁾ This property of porphyrin is excellent in catalyzing redox reactions.

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These systems are presently being studied in relation to oxygen reduction reactions, in particular the reactions occurring in fuel cells, and there has been significant progress in techniques for the incorporation of nitrogen into graphene-based catalysts. Pyridinic nitrogen on graphene sheets (Fig. 11) has been shown to catalyze efficiently the electrochemical oxygen reduction reaction. In this process, the pyridinic nitrogen modifies the system to generate a 4e⁻ reduction pathway via quaternary nitrogen formation in place of a 2e⁻ pathway. ^114,115) Thus, the atomic localization of nitrogen in the matrix is vital to obtaining and controlling electron donation to the reactants. In the case of N₂ plasma treatment of graphitic materials of carbon nanowall, electrical conductivity was increased by N doping. ¹¹⁶⁾ As described in Sect. 3.1, the endohedral N@C₆₀ encapsulate isolated electrons in the closed cage. The diamond NV center has emerged as a basic unit for quantum computing devices. Specifically, isolated electrons can act as either sources of free charges or as qubits having free spin. However, electronic or spintronic devices have not yet been realized based on this technology. Therefore, the doping of nitrogen atoms into various matrices with atomically localized control is necessary. Functional nitrogen can be provided only by atomically controlled doping based on the electron charge and spin. As a consequence, future research regarding the in situ doping of nitrogen will require interdisciplinary studies related to the development of new materials for device fabrication.

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The world's population will reach 8.6 billion in 2030, which will increase the demand for fertilizers, and so further developments in industrial nitrogen fixation processes are of great importance. ¹¹⁷⁾ It is well-known that the HB process plays a dominant role in chemical nitrogen fixation and is used to produce large amounts of ammonia. However, this is an energy-intensive process that consumes 1%–2% of the total energy used worldwide and 2% of the total natural gas. ¹¹⁷⁾ The HB process is also responsible for the emission of 300 million metric tons of CO₂ per year. Considering the fast-growing human population and the ongoing depletion of natural resources, it would be highly beneficial, both economically and environmentally, if the energy efficiency of nitrogen fixation could be improved using greener technology. As an example, Koga et al. demonstrated an environmentally friendly plasma technique for generating nitrogen-enriched organic manure. ¹¹⁸⁾ This was the first study in which the plasma was exposed on the soil to supplement soil with NH₄ ⁺ and NO₃ ⁻. However, other studies have focused on the synthesis of NH₃ or NO_x using plasma technology with different gas mixtures at various pressures. ¹⁸⁾ Earlier NH₃ production was reported using N₂ with H₂ ¹¹⁹⁾ or H₂O. ¹²⁰⁾ Plasma-catalytic processes can produce NH₃ at low atmospheric pressure with N₂/H₂ gas. ¹²¹⁾

Recently, NH₃ synthesis has been widely studied in plasma-electrochemical processes ¹²²⁾ and non-thermal plasmas using N₂ and H₂ gas at reduced pressure and low temperature. ¹²³⁾ For example, radio frequency excited (RF) plasma at low pressure (2 Pa) was used for NH₃ formation with and without tungsten or stainless steel surfaces using an N₂/H₂ gas mixture. ¹²⁴⁾ In another study, RF plasma with and without a furnace was used to produce NH₃ at low pressure (30–40 Pa), in the presence of a catalyst. ^125,126) The RF plasma caused the adsorption of NH_x and H radicals; in contrast, the microwave (MW) plasma caused the adsorption of N radicals. ¹²⁷⁾ It has been reported that RF and MW low-pressure plasma reactors cause a higher degree of vibrational excitation, which is useful for N₂ activation before the dissociation of N₂. ¹²³⁾

Many studies have previously reported the production of NO_x from air or N₂/O₂ mixtures using RF plasma, ¹²⁸⁾ MW plasma, ¹²⁹⁾ plasma jet, ¹³⁰⁾ DBD plasma, ¹³¹⁾ and gliding arc discharge. ¹³²⁾ The energy consumption of these methods varies widely, from 0.3 to 1600 MJ mol⁻¹. Mutel et al. synthesized NO using MW plasma at a pressure of 6600 Pa with a catalyst and N₂–O₂ gas mixture and the energy cost of this system was 0.084 MJ mol⁻¹. ¹³³⁾ The energy cost for NO production using the microwave plasma at a reduced pressure after applying a magnetic field was the lowest at 0.30 MJ mol⁻¹. ¹³⁴⁾ Although only plasma power was considered for low-value energy costs, the energy used for the cooling reactor was not included. On the other hand, gliding arc plasmas with N₂/O₂ gas mixtures have shown promising energy consumption rates of 3.6 MJ mol⁻¹ with 1.5 % NO_x yield. ¹³⁵⁾

The energy cost of production of NH₄NO₃, as well as that of NO, NO_x , and NH₃, is still higher with plasma technology than with existing technology. It is essential to shift towards plasma technology by introducing small-scale units to reduce environmental damage. In addition, plasma processes have the significant advantage of directly using abundant resources, such as air and water, and can be powered by electricity generated from renewable resources. There have been no reports of using direct plasma on the plant soil to enrich the soil with nitrogen species for later use in agriculture.

The Koga group has developed a low-pressure plasma capable of producing nitrogen-rich fertilizer without the use of expensive H₂ gas or a catalyst and has reported increased NH₄ ⁺ and NO₃ ⁻ concentrations in plasma-treated soil. Interestingly, the NO₃ ⁻ concentration in this material was higher than that in commercially available nitrogen-based fertilizer. The results revealed a 40% increase in early germination when using plasma-treated soil and a 34% increase in the shoot length of radish sprouts along with 39% higher sprout weights. The authors concluded that plasma-treated soil shows similar or better results with regard to the germination and growth of radish seeds compared with commercial nitrogen fertilizer.

The Koga group also demonstrated the effects of an atmospheric air plasma treatment on the germination and growth of radish seeds collected in two different harvest years with two different seed coat colors. ^136,137) Various treatments, including electromagnetic waves, ionizing radiation and atmospheric air plasma were used to accelerate the rate of seed germination, enhance plant growth and increase agricultural yields. ¹³⁸⁾ Recently, the use of atmospheric air plasma to treat seeds has increased. ¹³⁹⁾ In one study, the effects of RONS produced from an air scalar DBD plasma ¹⁴⁰⁾ were compared in terms of the germination rate and early growth of radish seeds harvested in two different years. In this study, each seed lot was sorted into two groups (brown and grey) and RONS-induced changes in the physical and chemical states, as well as the phytohormone and antioxidant levels in the radish seeds, were assessed before and after plasma treatment. ¹⁴⁰⁾ The average length of the brown seed sprouts was 24 ± 0.5 cm for the 2017 harvest, whereas, after the plasma treatment, the value was 20 ± 0.4 cm on the fourth day after treatment. The average length of the sprouts from brown seeds for the 2018 harvest was not changed significantly after plasma treatment. The authors concluded that RONS produced by an air DBD had a substantial effect on older seeds but not new seeds and that the seed coat color influenced the germination speed and sprout length.

In addition to air DBD, seeds treated with other plasmas have been found to exhibit enhanced germination and growth. ¹⁴¹⁾ Ito et al. reported that plasma-based agriculture can presently be divided into three categories. The first of these deals with plant seeds and bodies, while the second is water and nutrients containing short-lived species, and the last is the microbial environment in soil. ¹⁴²⁾ The electric field induces structural conformational changes in biomolecules, such as proteins. RNS participates in cell signaling pathways and mediates the biological responses to biotic and abiotic stress conditions in plants. The electromagnetic fields and RNS generated in plasma discharges can be used to directly stimulate the plant body and also to provide secondary effects by modifying water and soil. Yoshimura et al. reviewed the effects of the normothermic plasma irradiation of living organisms and reported that yeast cells must be irradiated at standard temperature. ¹⁴³⁾ Cells respond transiently to short-lived species such as ONOO⁻ anions and this transient response was found to promote the entry of calcium ions into cells. ¹⁴⁴⁾ However, the associated mechanisms have not been assessed comprehensively. The applications of plasmas in agriculture have been reviewed elsewhere ^145,146) and cover a wide range of research areas, including utilization during harvesting ⁴⁷⁾ and post-harvest ¹⁴⁷⁾ as well as in the food supply chains. ¹⁴⁸⁾

The biological processes of living organisms involve complex interactions between many signaling molecules. The balance between the intrinsic production and consumption of signaling species and external perturbation affects physiological homeostasis ¹⁴⁹⁾ and RNS function as major regulators of various signaling pathways. ¹⁵⁰⁾ For example, NO and ONOO⁻ are known to play important roles in cell survival and death processes by regulating mitochondrial transmembrane permeability. ^151,152) Intracellular and intercellular systems are based on many molecular compounds, proteins and signaling pathways. That is, biological functions do not stem from a single agent, but from collective interactions among components of a network. Network biology is therefore a systematic approach to understanding how these agents and their interactions trigger specific functions. ^153,154) Network analysis based on mathematical graph theory has been applied in various fields, including physics, chemistry, biology, computer science, geography, economics and sociology. ^154–156) In graph theory, the elements (reagents) of the system and the interactions between these elements (reaction pathways) are represented as nodes and edges, respectively, to model pairwise relationships. Therefore, a graphical and theoretical analysis can capture not only the unique characteristics of a particular network but also the statistical characteristics common to a wide range of networks.

Both gas phase and liquid phase plasma-induced chemistry are sufficiently complex to form network structures. Recently, the basic characteristics of gas-phase chemical networks have been examined. ^157–161) Sakai et al. applied graph theory to the study of CH₄ plasma and SiH₄ plasma chemistry to identify the species that play a central role in triggering subsequent reactions. They showed that the graph theory approach allows rapid visualization and classification of these complex chemical systems. ^142,157) In addition, Sakai's group elucidated the growth mechanism associated with the SiH₄ plasma reaction network. ¹⁵⁹⁾ Holmes et al. also used graph theory and proposed an algorithm to quantify the requirements for the target's chemical composition. ¹⁶¹⁾

Murakami and Sakai extracted essential information from the complex reaction data obtained for a He + humid air plasma ^162–164) and devised a method to rescale the reaction network based on graph theory analysis. Furthermore, they quantified the impact of network rescaling on a quasi-2D numerical simulation of a plasma jet. Figure 12 shows the topological characteristics of the chemistry within a plasma obtained from a He + N₂ + O₂ + H₂O + CO₂ mixture, together with the relationship between closeness centrality and betweenness centrality, and a network diagram of closeness centrality. ¹⁶⁰⁾ Closeness centrality is a measure of how quickly information spreads from one particular species in the network to other reachable species, and so those species with high closeness centrality are placed in the center of the network. Betweenness centrality represents the controllability, informativeness or importance of an intermediate that directly or indirectly bridges reactants and products. Figure 12 shows that oxygen molecules and helium atoms, acting as the primary background species, and electrons, acting as the reaction driver species, dominate the network topology. Ground state atomic nitrogen N (⁴S), NO₂ and ·OH are also in the most central region because these species have relatively high betweenness centrality. Species with higher betweenness centrality interact with a wider range of other species, more intermediate reactions, and integrate the entire reaction system.

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During the biological and agricultural applications of atmospheric air plasmas, various reactive species are generated and applied to biological targets. Through a reaction network analysis, it is possible to predict the trajectory of functional nitrogen while obtaining an overview of the more general framework of the plasma-biology interactions.

As an interdisciplinary numerical study of the interactions between plasma chemistry and molecular biology, Murakami developed a biochemical network model of mitochondrial redox homeostasis and energy metabolism. This work clarified the effects of reactive species from the plasma on the intracellular functions through a time-dependent 0D numerical simulation. ¹⁶⁵⁾ The study was motivated by recent experimental results showing that nitrogen-based plasma lowers mitochondrial membrane potentials and significantly affects cell survival rates. ¹⁶⁶⁾ Figure 13 shows a pathway map and a topology diagram of the modeled biochemical network. ¹⁶⁵⁾ The numerical model includes fundamental mitochondrial functions associated with pyruvic acid oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation involving the respiratory chain, adenosine triphosphate/adenosine diphosphate (ATP/ADP) synthesis machinery, and ROS/RNS-mediated mechanisms. The functions of the cell membrane, cytosol and glycolysis are not considered. The effects of plasma irradiation are modeled based on the influx of H₂O₂ to an ROS regulation system and the change in mitochondrial transmembrane potential induced by the effect of NO/ONOO⁻ on membrane permeability. Nicotinamide adenine dinucleotide (NAD; C₂₁H₂₇N₇O₁₄P₂), its reduced form NADH and oxidized form NAD⁺ play a central role in oxidative phosphorylation in aerobic organisms. This work has demonstrated that the plasma-derived RONS stimuli greatly affect ROS oscillation, NAD redox and ATP/ADP conversion. This plasma-induced ROS influx changes the intrinsic intramitochondrial redox oscillatory rhythm from homeostatic to irreversible. It is also evident that the RNS-driven change in transmembrane permeability controls the redox regulation system and ATP/ADP metabolism through NAD-mediated reactions, that is the RNS-induced deterioration of the mitochondrial transmembrane potential promotes the ATP → ADP transformation.

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We examine recent progress in research related to RNS in plant defense and phytohormones as signaling molecules and post-translational modification. The effects of RNS can be understood based on molecular modifications. Prior studies have focused on plant defense systems ¹⁶⁷⁾ and have found that S-nitrosylation (the attachment of NO to a cysteine thiol moiety) triggers the release of phytohormones related to seed germination and seedling growth. ¹⁶⁸⁾

One of the basic responses of plants is defense against pathogens. In the event of infection, the plant encloses the pathogen in a limited area and responds with resistance. For this response to occur, the plant must recognize the pathogens, generate a signal and transmit this signal to the surrounding area, which involves microbial substances known as elicitors. Genes related to defense responses are expressed and cells in the enclosed region containing the pathogens are killed through hypersensitive cell death. As well, plants produce pathogenesis-related proteins and phytoalexins, which are low molecular weight antibacterial substances. It is known that ROS and RNS, in particular O₂ ⁻, NO and ONOO⁻, act as signaling molecules in these systems, and that hypersensitive cell death is induced by elicitors produced from NO, O₂ and H₂O₂.

Monjil et al. found that a synthetic bis-aryl-methanone compound (NUBS-4190) and a methanol extract from a pathogen (the mycelia of P. infestans) that contained lipophilic compounds were elicited by potato leaves in response to NO. ^169,170) The gene for Arabidopsis thaliana nitric oxide-associated 1 (AtNOA1) has been identified as a putative regulator of NOS activity in plants. ¹⁷¹⁾ In N. benthamiana leaves, the pathogenesis-related 1a (NbPR1a) gene and nitric oxide associated 1 (NbNOA1) gene, a homolog of AtNOA1, were found to be associated with infection resistance. ^172,173) Moreover, nitrate reductase (NR) has been shown to be responsible in part for INF1 elicitor-induced NO production. ^174,175) Protein S-nitrosylation, a redox-related modification of cysteine thiol groups by NO, is known to be one of the most important post-translational modifications regulating the activity and interactions of proteins. The proteomic analysis of S-nitrosylated proteins in potato (Solanum tuberosum) using a modified biotin switch assay found that dehydroascorbate reductase 1 (DHAR1, EC 1.8.5.1) is modified by S-nitrosylation, inducing glutathione-dependent dehydroascorbate-reducing activity. ¹⁷⁶⁾ In addition, intracellular fluorescent imaging of ONOO⁻ using aminophenyl fluorescein by Saito et al. identified tyrosine nitration in the defense responses of plants and showed that ONOO⁻ reacts with tyrosine residues in proteins to form nitrotyrosine in tobacco BY-2 cells treated with INF1. ¹⁷⁷⁾ ONOO⁻ seems to indirectly mediate the nitration of proteins and nucleic acids or phenolics. ¹⁷⁸⁾ The oxidative and nitrative modifications of proteins may also regulate some biological functions by evolving physiologically adaptive responses.

Kawakita et al. demonstrated that NO production is involved in plant-microbe interactions and is an effective invasion strategy for pathogens but is also essential for plant defense responses ¹⁷⁹⁾ (Fig. 14). Systematic acquired resistance in plants is regulated by the non-expression of pathogenesis-related gene I (NPR1), which forms oligomers via S-nitrosylation (indicated by –SNO in Fig. 14) based on the NO concentration. Salicylic acid (SA) dissociates the NPR1 oligomers (forming the thiol moiety indicated by –SH in Fig. 14) that translocate to provide nuclei and gene expressions involved with plant defense genes. Peroxiredoxin (PrxII) is a reductase for ONOO⁻ and this enzymatic activity is regulated by S-nitrosylation. Therefore, superoxide radicals (O₂ ⁻) generated by respiratory burst oxidase homolog D (RBOHD) play an important role in generating ONOO⁻. Thus, the cellular signaling of NO is delivered and cascades through multiple molecular modification pathways in the plant defense system.

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The nitration of tyrosine is a key topic in biochemistry related to the oxidative modification of proteins. ¹⁸⁰⁾ This reaction is mediated by ONOO⁻ via the substitution of a nitro group for a hydrogen atom. ¹⁸¹⁾ As an example, in cytochromes iron sites cooperate to form ferrous superoxo (Fe^IIOO) and ferric nitrosyl (Fe^IIINO) intermediates. ¹⁸²⁾ These ONOO⁻ intermediates dissociate homolytically into O₂ ⁻ and NO. ¹⁸²⁾ Lastly, biosynthetic gene clusters that function to promote the nitrosation of metalloenzymes are found in bacteria. ¹⁸³⁾ These metalloenzymes catalyze the oxidative rearrangement of the guanidine group. ¹⁸³⁾ NO production in mammalian cells is catalyzed by NO synthase (NOS), although no NOS-like enzymes have been identified in plants. ¹⁸⁴⁾ Even so, the actions of RNS with regard to NO metabolism, signaling and stress response are important.

In addition to tyrosine nitration, these post-translational modifications are considered to be essential biological responses related to the collaborative effects of NO and O₂ ⁻ radicals as well as ONOO⁻ at the molecular level. Diazo (R–N=N), nitro (R–NO₂) and nitrosyl (R–N=O) moieties are important. Diazo moieties have been shown to give rise to various reactive intermediates, including vinyl radicals, o-quinone methide (C₇H₆O), acylfulvene (C₁₄H₁₆O₂), and a covalent adduct resulting from both reductive and nucleophilic activation of diazo groups. ¹⁸⁵⁾ Carbonyl groups and neighboring methylidene or hydroxyl groups work together to form diazo groups by combining with reactants such as ONOO⁻. In addition, the quinone moiety may undergo redox cycling to produce ROS. ^186,187) [Figure 14(b)] Once hydroxyl groups are oxidized by dehydration and dehydrogenation forming radical, ONOO⁻ reacts with the radical forming nitro moiety and O₂ ⁻ radical. This nitration plays a pivotal role in post-translational modifications. Although it has not yet been completely proven, it has been hypothesized that the RNS generated in plasma, involving NO, NO₂ ⁻, and ONOO⁻, may directly and indirectly affect the biochemical processes of multiple cell signaling and communication pathways.

Phytohormones control and regulate plant growth and development by acting as signal molecules produced within plants at extremely low concentrations. ¹⁸⁸⁾ These compounds include abscisic acid (ABA), auxin (OC), gibberellin (GA), jasmonic acid (JA), SA, cytokinins, strigolactone, ethylene, peptides and polyamines. As a result of recent advances in functional genomics, the metabolism of ABA (a plant hormone that regulates seed dormancy and germination) involving biosynthesis and catabolism has been widely elucidated. ^189,190) Figure 15 shows the mechanisms of the regulation of dormancy attenuation and germination promotion of seeds. ABA levels promote seed dormancy and germination; during seed germination, ABA levels decrease and GA levels increase. To attenuate this dormancy level, NO-related metabolism in relation to ABA contributes to the promotion of seed germination. In response to internal and external cues, seeds undergo changes in ABA levels, and large-scale comparative studies have been conducted on seeds under different environmental conditions. ^191,192) The variants of this compound include ABA1 and ABA2 acting as protein phosphatases, and ABA3, ABA4 and ABA5, all of which are related to plant dormancy. ABA4 also plays a role in response to NO₂ and NH₃. ¹⁹³⁾ Using two ABA metabolism mutants, an ABA-deficient mutant (ABA2) and an ABA over-accumulation mutant (CYP707A1A2A3 triple mutant), Okamoto et al. studied transcriptomes in seeds for endogenous ABA. ¹⁹⁴⁾ Yamagishi et al. reported that the CHO1 mutation acts as a strong enhancer of the ABI5 mutant, whereas the CHO1 ABI4 double mutant shows ABA resistance similar to the ABI4 single mutant. ¹⁹⁵⁾

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Similar to lettuce seeds, the germination of Lactuca sativa seeds is regulated by alterations in the levels of phytohormones, such as ABA and GA. ¹⁹⁶⁾ The imbibition responses of Arabidopsis seeds were analyzed by acquiring transcriptomic and hormone profiles. ¹⁹⁷⁾ In the case of dormant Arabidopsis seeds, nitrate levels were reduced but exogenous nitrate concentrations increased with ABA uptake by the seeds. The expression of the ABA catabolic gene CYP707A2 was also regulated by exogenous nitrate. In addition, the CYP707A2–1 mutant failed to reduce seed ABA levels in response to either endogenous or exogenous nitrate. ¹⁹⁸⁾ The ABA level in seeds evidently controls germination and seedling growth throughout the expression of the gene for the leucine zipper transcriptional factor ABI5, with is counteracted by NO. ABI5 is therefore accumulated in conjunction with impaired NO homeostasis. The S-nitrosylation of ABI5 at cysteine-153 facilitates its degradation through CULLIN4-based and KEEP ON GOING E3 ligases and promotes seed germination. Conversely, the mutation of ABI5 at cysteine-153 deregulates protein stability and the inhibition of seed germination by NO depletion. An inverse molecular link between NO and ABA hormone signaling through distinct posttranslational modifications of ABI5 during early seedling development has also been identified. ¹⁶⁸⁾ Hypothetically, plasma-generated RNS may contribute to the regulation of the ABA signaling network by promoting post-translational modifications, such as tyrosine nitration, S-nitrosylation. Notably, extracellular NO gas is known to be affected exogenously, which explains why intracellular NO acting as the signaling molecule should be regulated at the desired location inside a cell. It has also attracted the plasma-based RNS, which is clearly recognized as an intracellular signaling molecule; however, the mechanisms underlying the regulation of NO have not yet been elucidated.

In plant science, genomic screening using gene mutants is often employed for dormant germination control. The mutation of genes is possible by base transformation using the reactions of specific compounds. Gene-editing techniques such as CRISPR/Cas9 are also used and an experimental system to quantitatively measure the effects of RNS on living organisms (that is, plants) can be constructed. The comprehensive disruption of candidate genes using genome editing methods has shown that genetic mutations may be involved in the effects of RNS. Screening of the mutant libraries can also identify genes that have been found to have physiological effects. A compound library can be screened to identify drugs that have been shown to have these effects, and the identification of molecules involved in this process can proceed in combination with the selection of mutants with different sensitivities to these compounds.

In chemical biology in contrast to genetic studies, the screening of bioactive compounds produced by RNS can be used to elucidate target molecules and signaling pathways. A comprehensive analysis of the nitrosylation (–NO) and nitration (–NO₂) of amino acid residues after the translation of gene expressions is required, using post-translational modification omics (PTM-omics). This technique can assist in elucidating the effects of NO and NO₂ on proteasome and gene transcription regulation.

In biochemistry, the relationship between metabolome and phenotypes involving epigenetic and post-translational modifications is an important area of study. There are so-called direct effects of RNS compounds on seeds and plants through biological transients and indirect effects through plant physiological substances. ABI5 was first identified as a transcriptional regulator of ABA related to NO, based on the symbiotic expression of serine from cysteine. ABI5 mutants exhibit S-nitrosylation, in which the SH group of the cysteine residue binds with NO, and this reaction is accompanied by oxygen metabolism and ubiquitination.

In plant immunity, Ochi et al. demonstrated that the plasma irradiation of water-soaked rice seeds stimulated the generation of ROS that were correlated with the suppression of plant disease. ¹⁹⁹⁾ Both RNS and ROS function as important signaling factors in plant immunity and it is assumed that the RNS produced by plasma may act directly or indirectly on plant immune mechanisms. ²⁰⁰⁾ Therefore, the involvement of plasma-derived RNS in signal transduction due to pathogen infection and the epigenetic regulation of plant immune mechanisms should be analyzed. A recent study revealed that irradiation of Arabidopsis thaliana with different active species, including plasma-derived RNS, selectively activated the expression of marker genes for defense responses.

In the context of plasma-based agriculture, the development of RNS doping techniques, the effects on biological materials, and the lifecycle phase in which functionalization is achieved are all important. Possible reaction pathways may involve receptors, post-translational modification, proteolysis control with poly-ubiquitination and proteasome, or regulation of gene expression with the nitration of nucleic acids (Fig. 16). The effects of RNS are distinguished by primary and secondary actions. In the simplest case, RNS directly regulate biological responses. However, we should consider that the secondary effects of the nitrosylation and nitration of amino acid residues and proteolysis control also regulate the biological responses of RNS. The timing of RNS doping of the cell may also be important for obtaining functional responses.

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The previous sections examined the transportation of nitrogen in living systems and the hierarchical analysis of spatiotemporal phenomena associated with RNS, as illustrated in Fig. 17. Table IV lists the representative results of the in-situ measurements of plasma processing. In this process, nitrogen compounds are synthesized from air and electricity. As an example, RNS in the gas phase are transported into aqueous or solid phases via boundaries. In the first case, solid-state materials can be functionalized by the doping of nitrogen into the bulk matrices of inorganic substances such as catalysts and ceramics, or of organics such as polymers. In this manner, in situ plasma and ion irradiation can provide nitrogen doping to create new functionalities without the use of ammonia or acid.

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At present, we do not have an atomically resolved understanding of the mechanism related to the localization of nitrogen during in situ nitrogen doping. Thus, advancements in techniques to measure and analyze the transportation of various species are required. Spatiotemporal and simultaneous observations of multiple species at various points should be conducted.

7.1.1. Electron behavior

7.1.2. Transportation of reactive species in the gas phase

7.1.3. Transportation of reactive species in the aqueous phase

7.1.4. Atomic localization of functional nitrogen

Before the suggested development of in situ utilization of nitrogen, i.e. the functional nitrogen method, one problem is the environmental issues responsible for residual risks. Conventional methods that use long-lived substances, such as drugs and fertilizers, are raised on environmental issues, and thus we should consider reuse, recycling, and recovery; otherwise, discarding forms of pollution and contamination. This is due to long-lived species. Indeed, the impact of residual pesticides on the biosphere and chemical fertilizers pollutes rivers and lakes to indirectly impact ecosystems. To solve these problems, we propose the use of short-lived species precisely at optimal locations and at suitable timing. A variety of short-lived species, particularly RNS generated in plasma, can be potentially used in many applications. In this view, it is advantageous that an environmentally benign method, using only air, water, and electricity, can be offered by the plasma-based in situ functionalization of RNS. To realize this, we should scientifically solve the responses seen in materials and living organisms, with respect to unstable nitrogen dosages. In dose dependences, it is common for the optimum dose to appear. (Fig. 18) The functionalization depends on the dose with optima. Therefore, there are several technological problems that need to be solved, including ways to control the localization of atomic nitrogen, gain the functionality of localized nitrogen, and stimulate living organisms using plasma-based RNS.

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Next, the atomic localization of nitrogen in the lattice system presents a problem. The localization of atomic nitrogen to provide various functions is a common process in both materials science and living systems. One example is the formation of NV centers in diamond and nitrogen atoms having isolated electronic spins in fullerene, which can function as qubits. The fabrication of these qubits must not damage the diamond crystal matrix or the substrate material during the localization of the atomic nitrogen, because small fluctuations in the periodic potential of the crystal generate spatially optimal positions with energy minima. This process is analogous to the doping of semiconductor devices.

The irreversible processes associated with the doping of living systems or inanimate materials are associated with the lifecycles of these substances (Fig. 19). Various events such as fracture, damage and fatigue must be included when assessing a lifecycle, and phenomena such as senescence, healing and disease are also important in biological systems. For example, in each phase of the lifecycle, the hierarchically trans-scale representation of the atomic localization of nitrogen and the network of observed parameters with hidden states can be examined to bridge between macroscopic and microscopic dynamics.

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The plant lifecycle stages include seed germination, seedling, plant growth, mature plant and flower, and fruit with seed. (Fig. 19) In each phase, the plant uptakes and assimilates nitrogen as a nutrient. However, the timing of the nitrogen uptake and any effect of environmental RNS on the plant assimilation process has not yet been clarified. During the plasma activation of seeds and plants, the timing and duration of exposure to the RNS are certainly essential. That is, the network controls the functionalization of RNS during plasma processing.

In summary, we encompass an outlook for the future. (Fig. 20) Conventional processing is designed by the application of plasma treatments to both materials and living organisms. This is regarded as a linear cascade from plasma generation to process outcomes, which has previously been proven by many innovative emerging technologies. However, as the linear cascade model of plasma effects has been developed, it is necessary to increase our understanding of the constituents and compositions of the plasma-generated species, and the individual effects of each species need to be deconvolved. Moreover, it has been revealed to be essential to gain the plasma effects through the evolution of multiple pathways of reactions and synergisms of multiple species. In other words, the present research has already realized the preparation of the selected reactive species and decomposes the complex reaction paths into its primary and secondary impacts on hierarchical systems, such as living organisms, and to reveal hidden or latent reactions. Based on this knowledge, we may understand the relationship between the selected reactive species and the emergence of functions in materials and living organisms. This is insufficient to engineer the functionality of the reactive species. Since we should analyze all the multiple pathways quantitatively, we emphasize the requirement for multiple in situ and real-time measurements during this process.

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As a consequence, our outlook for the future is the sciences of the clustering networks that are connected in a complicated manner among plasma, liquid, solid, and living organisms. An ensemble of multiple states is analyzed. For either materials or living organisms, changes remain in the irreversible life cycle. Similar to plasma stimulation, it is believed that the system responds by modifying the connectivity of the clustering networks. In other words, the stimulated states of the network are temporarily relaxed by losing their excited energy. This relaxation process can be decomposed into an ensemble of kinetically controlled reactions on the network. Lastly, a trans-scale understanding of the organized network state at each phase of the lifecycle would be possible by a series of in situ real-time measurements of multiple species with spatiotemporal resolutions (Fig. 19 bottom).