Direct and Indirect Chemiluminescence: Reactions, Mechanisms and Challenges

Emission of light by matter can occur through a variety of mechanisms. When it results from an electronically excited state of a species produced by a chemical reaction, it is called chemiluminescence (CL). The phenomenon can take place both in natural and artificial chemical systems and it has been utilized in a variety of applications. In this review, we aim to revisit some of the latest CL applications based on direct and indirect production modes. The characteristics of the chemical reactions and the underpinning CL mechanisms are thoroughly discussed in view of studies from the very recent bibliography. Different methodologies aiming at higher CL efficiencies are summarized and presented in detail, including CL type and scaffolds used in each study. The CL role in the development of efficient therapeutic platforms is also discussed in relation to the Reactive Oxygen Species (ROS) and singlet oxygen (1O2) produced, as final products. Moreover, recent research results from our team are included regarding the behavior of commonly used photosensitizers upon chemical activation under CL conditions. The CL prospects in imaging, biomimetic organic and radical chemistry, and therapeutics are critically presented in respect to the persisting challenges and limitations of the existing strategies to date.


Introduction
Chemiluminescence (CL) is the spontaneous emission of light from an electronically excited state of a species produced by a chemical reaction [1]. Nature uses CL (called bioluminescence) in many living organisms such as fireflies, mushrooms, shells, jellyfish, [2] worms and bacteria, mainly for communication or defense roles [3]. In biological systems, bioluminescence occurs from in situ enzyme-catalyzed chemical transformations, for example luciferin [4] reacts with oxygen in the presence of luciferase enzyme [5], magnesium [6][7][8][9] or calcium ions and adenosine triphosphate (ATP), leading to luminescence [10][11][12]. The reaction involves chemical activation of specific molecules (A) via oxidation, resulting in a chemiexcited intermediate (CEI) (C* or D*; excited species indicated by *) that releases its energy either via light emission (direct) or by transferring it, through a chemiluminescence resonance energy transfer (CRET) process, to an adjacent fluorophore (E) that becomes excited (E*); this fluorophore subsequently releases part of its energy by emitting light. These two distinct mechanisms dictate the two types of CL, direct and indirect, respectively, as shown in Figure 1. Typical examples of molecules that emit light via direct CL are luminol and luc two of the most well-studied luminogenic substances which are susceptible to oxi by hydrogen peroxide (H2O2) and superoxide radical anion (O2 •-). The emitted ligh result of changes in the chemical structure of the produced chemiexcited interme Under certain conditions, singlet oxygen ( 1 O2) can also be produced at high amou chemiexcitation product, by peroxides via direct CL. 1 O2 upon radiative relaxation infrared light at 1270 nm if not previously trapped, affording oxidation products [1 production of 1 O2 under CL conditions is discussed later in the text (vide infra). In ad oxalate esters and 1,2-dioxetane derivates serve as efficient precursors both for dire indirect CL providing tunable emission systems depending on the photophysical p ties of the energy acceptor fluorophore, with a wide range of applications in sensin diagnosis, molecular imaging, and cancer treatment [14,15].
In indirect CL, energy transfer takes place, resulting in excitation of a fluoroph a photosensitizer that can further act independently. In the presence of molecular o (O2), the photosensitizer can generate reactive oxygen species (ROS) such as O2 •-, hy radicals (HO • ) or 1 O2. These species have a broad spectrum of reactivity with biomol [16][17][18][19] and can be used in photodynamic therapy (PDT) to induce selectively canc death, as well as other chemical transformations [20][21][22][23].
Most of the studied CL systems exhibit flash-type single-color light emission usually limit their applications. Long-lasting multicolor CL in aqueous solutions is desirable, especially in biological applications, however a controllable chemical proc still remains a challenge. CL is a powerful tool in analytical chemistry that is used detection and quantification of reactive oxygen and nitrogen species [24,25], and n ous biological materials such as DNA [26], RNA [27], proteins [28], microorganisms metals [14,29], etc. [30,31]. Lately, immunoassays have been also used for the detec SARS-CoV-2 [32].
Moreover, CL has been considered as an alternative photo-uncaging option in delivery technologies for meeting the increasing demand for efficient strategies [15] ever, in biological systems, several issues have to be addressed, such as: (i) the tissu etration of the incident light used for photoactivation of the emitted photons repr one of major barriers limiting efficiency in imaging or other applications, (ii) light scattered or absorbed by other biomolecules inside the cell, and (iii) light intensity versely proportional to the square of the distance from the light source, in this way ing weak interaction between an incident photon and a molecule (see Jablonski e diagram, Figure 2) which leads to poor photochemical interactions. CL has increa attracted the interest of the research community, providing solutions to persisting lems of typical photochemical procedures. Recent successful examples from imagin therapeutics fields highlight the great potential of CL-induced applications. To da reported reviews discuss CL mainly focused on chemiluminescent molecules such minol, cypridina luciferin and peroxyoxalates or analogues that can be used in se imagine and therapeutic applications [33][34][35][36]. Typical examples of molecules that emit light via direct CL are luminol and luciferin, two of the most well-studied luminogenic substances which are susceptible to oxidation by hydrogen peroxide (H 2 O 2 ) and superoxide radical anion (O 2 •-). The emitted light is the result of changes in the chemical structure of the produced chemiexcited intermediates. Under certain conditions, singlet oxygen ( 1 O 2 ) can also be produced at high amounts, as chemiexcitation product, by peroxides via direct CL. 1 O 2 upon radiative relaxation emits infrared light at 1270 nm if not previously trapped, affording oxidation products [13]. The production of 1 O 2 under CL conditions is discussed later in the text (vide infra). In addition, oxalate esters and 1,2-dioxetane derivates serve as efficient precursors both for direct and indirect CL providing tunable emission systems depending on the photophysical properties of the energy acceptor fluorophore, with a wide range of applications in sensing and diagnosis, molecular imaging, and cancer treatment [14,15].
In indirect CL, energy transfer takes place, resulting in excitation of a fluorophore or a photosensitizer that can further act independently. In the presence of molecular oxygen (O 2 ), the photosensitizer can generate reactive oxygen species (ROS) such as O 2 •-, hydroxyl radicals (HO • ) or 1 O 2 . These species have a broad spectrum of reactivity with biomolecules [16][17][18][19] and can be used in photodynamic therapy (PDT) to induce selectively cancer cell death, as well as other chemical transformations [20][21][22][23].
Most of the studied CL systems exhibit flash-type single-color light emissions that usually limit their applications. Long-lasting multicolor CL in aqueous solutions is highly desirable, especially in biological applications, however a controllable chemical procedure still remains a challenge. CL is a powerful tool in analytical chemistry that is used for the detection and quantification of reactive oxygen and nitrogen species [24,25], and numerous biological materials such as DNA [26], RNA [27], proteins [28], microorganisms, cells, metals [14,29], etc. [30,31]. Lately, immunoassays have been also used for the detection of SARS-CoV-2 [32].
Moreover, CL has been considered as an alternative photo-uncaging option in drugdelivery technologies for meeting the increasing demand for efficient strategies [15]. However, in biological systems, several issues have to be addressed, such as: (i) the tissue penetration of the incident light used for photoactivation of the emitted photons represents one of major barriers limiting efficiency in imaging or other applications, (ii) light can be scattered or absorbed by other biomolecules inside the cell, and (iii) light intensity is inversely proportional to the square of the distance from the light source, in this way causing weak interaction between an incident photon and a molecule (see Jablonski energy diagram, Figure 2) which leads to poor photochemical interactions. CL has increasingly attracted the interest of the research community, providing solutions to persisting problems of typical photochemical procedures. Recent successful examples from imaging and therapeutics fields highlight the great potential of CL-induced applications. To date, the reported reviews discuss CL mainly focused on chemiluminescent molecules such as luminol, cypridina luciferin and peroxyoxalates or analogues that can be used in sensing, imagine and therapeutic applications [33][34][35][36].
Upon absorption of a photon of a particular energy, the molecule becom to an electronically excited state ( Figure 2). Among the various paths tha to dissipate the excess energy, radiative relaxation can occur via phospho or fluorescence (S1→S0). Electrochemiluminescence (ECL) is the production of light by an ex has been generated electrochemically, i.e., an electron transfer reaction at electrode (Figure 3). Numerous review articles and monographs are foun to date on the electrogenerated CL, describing the fundamentals, mecha cations. Thus, in this review we will not further discuss this type of CL [ The aim of this review is to provide a summary of the recent developments in this field, based on direct and indirect CL. An overview of the strategies leading to better analytical sensitivity is included along with recent indicative examples. The role of 1 O 2 as a chemiexcitation product is also described in relation to the application and further development of more efficient CL-induced therapeutic platforms. Finally, unpublished results of our research regarding the behavior of commonly used photosensitizers as energy acceptors under CL conditions are also included together with discussion on the prospects and the challenges of CL applications in imaging, biomimetic organic and radical chemistry, and therapeutics.

Mechanistic Aspects of the Types of Chemiluminescence: Direct and Indirect
Among the different types of luminescence, the most typical ones are photoluminescence (PL), chemiluminescence (CL), bioluminescence (BL), and electrochemiluminescence (ECL). A list of recent references for each luminescence type is provided in Table 1. Electrochemical Excitation [42] In PL, incident photons are the driving force for the emission of light by a molecule. Upon absorption of a photon of a particular energy, the molecule becomes photoexcited to an electronically excited state ( Figure 2). Among the various paths that can be followed to dissipate the excess energy, radiative relaxation can occur via phosphorescence (T 1 →S 0 ) or fluorescence (S 1 →S 0 ). Electrochemiluminescence (ECL) is the production of light by an excited species that has been generated electrochemically, i.e., an electron transfer reaction at the surface of an electrode (Figure 3). Numerous review articles and monographs are found in the literature to date on the electrogenerated CL, describing the fundamentals, mechanisms and applications. Thus, in this review we will not further discuss this type of CL [42].  In bioluminescence (BL), which is naturally occurring CL, light i ganisms. Various luminophore molecules, enzymes and cofactors more than 30 different bioluminescent systems known to date [40]. before, CL is a process in which chemical reactions can generate an e who can emit light directly-direct emission-or transfer its energy t ophore-indirect emission- (Figure 1). The FRET (Förster Resonance Fluorescence Resonance Energy Transfer), which refers to the nonradi ergy from an excited donor to an acceptor mediated by electronic dipo between the oxidized CL reagent (excited state donor) and a fluoroph ferred as chemiluminescence resonance energy transfer (CRET). Apa and sensing applications, recent advances in the PDT field revealed t an alternative to conventional light sources. The proximity of the ligh get" molecule, as is the case for CL, assists the photochemical transfor faster and photon flux requirements are lower in respect to the proced ventional type of light sources [41].

Direct Chemiluminescence
To the best of our knowledge, one of the very first examples o back in 1928 [43], and it is based on the oxidation of luminol by H2O2 After luminol's oxidation, a strong blue emission with λmax = 425 nm a reverse intersystem crossing (rISC) [46][47][48] that can last from a seco depending on the quantity of the reacting species, the presence of sp the "feeding" process. The light emission of luminol and its derivative catalysts such as peroxidase and heme, which are usually used as add horseradish peroxidase (HRP) is involved in the production of a lum two molecules of luminol anion [49]. In bioluminescence (BL), which is naturally occurring CL, light is emitted by live organisms. Various luminophore molecules, enzymes and cofactors are involved in the more than 30 different bioluminescent systems known to date [40]. As was mentioned before, CL is a process in which chemical reactions can generate an excited intermediate who can emit light directly-direct emission-or transfer its energy to an acceptor fluorophore-indirect emission- (Figure 1). The FRET (Förster Resonance Energy Transfer or Fluorescence Resonance Energy Transfer), which refers to the nonradiative transfer of energy from an excited donor to an acceptor mediated by electronic dipole-dipole coupling, between the oxidized CL reagent (excited state donor) and a fluorophore (acceptor) is referred as chemiluminescence resonance energy transfer (CRET). Apart from bioimaging and sensing applications, recent advances in the PDT field revealed that CL could act as an alternative to conventional light sources. The proximity of the light source to the "target" molecule, as is the case for CL, assists the photochemical transformations to progress faster and photon flux requirements are lower in respect to the procedures based on conventional type of light sources [41].

Direct Chemiluminescence
To the best of our knowledge, one of the very first examples of direct CL is dated back in 1928 [43], and it is based on the oxidation of luminol by H 2 O 2 (Scheme 1) [44,45]. After luminol's oxidation, a strong blue emission with λ max = 425 nm is recorded through a reverse intersystem crossing (rISC) [46][47][48] that can last from a second up to few hours, depending on the quantity of the reacting species, the presence of specific additives and the "feeding" process. The light emission of luminol and its derivatives can be assisted by catalysts such as peroxidase and heme, which are usually used as additives. For example, horseradish peroxidase (HRP) is involved in the production of a luminol free radical by two molecules of luminol anion [49]. In bioluminescence (BL), which is naturally occurring CL, light is emitted by live organisms. Various luminophore molecules, enzymes and cofactors are involved in the more than 30 different bioluminescent systems known to date [40]. As was mentioned before, CL is a process in which chemical reactions can generate an excited intermediate who can emit light directly-direct emission-or transfer its energy to an acceptor fluorophore-indirect emission- (Figure 1). The FRET (Förster Resonance Energy Transfer or Fluorescence Resonance Energy Transfer), which refers to the nonradiative transfer of energy from an excited donor to an acceptor mediated by electronic dipole-dipole coupling, between the oxidized CL reagent (excited state donor) and a fluorophore (acceptor) is referred as chemiluminescence resonance energy transfer (CRET). Apart from bioimaging and sensing applications, recent advances in the PDT field revealed that CL could act as an alternative to conventional light sources. The proximity of the light source to the "target" molecule, as is the case for CL, assists the photochemical transformations to progress faster and photon flux requirements are lower in respect to the procedures based on conventional type of light sources [41].

Direct Chemiluminescence
To the best of our knowledge, one of the very first examples of direct CL is dated back in 1928 [43], and it is based on the oxidation of luminol by H2O2 (Scheme 1) [44,45]. After luminol's oxidation, a strong blue emission with λmax = 425 nm is recorded through a reverse intersystem crossing (rISC) [46][47][48] that can last from a second up to few hours, depending on the quantity of the reacting species, the presence of specific additives and the "feeding" process. The light emission of luminol and its derivatives can be assisted by catalysts such as peroxidase and heme, which are usually used as additives. For example, horseradish peroxidase (HRP) is involved in the production of a luminol free radical by two molecules of luminol anion [49]. Similarly to luminol, cypridina luciferin emits blue light after oxidation by molecular oxygen catalyzed by cypridina luciferase (Scheme 2) [50][51][52]. Moreover, emission was also recorded after oxidation of 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, a cypridina luciferin analogue, by O2 •-. At the time of its discovery, this selective oxidation  Similarly to luminol, cypridina luciferin emits blue light after oxidation by molecular oxygen catalyzed by cypridina luciferase (Scheme 2) [50][51][52]. Moreover, emission was also recorded after oxidation of 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, a cypridina luciferin analogue, by O 2 •-. At the time of its discovery, this selective oxidation provided a unique opportunity for direct CL detection of O 2 •and further development of other similar chemiluminescent probes [6].
Molecules 2021, 26, 7664 5 provided a unique opportunity for direct CL detection of O2 •-and further develop of other similar chemiluminescent probes [6].
Scheme 2. CL of cypridina luciferin, coelenterazine and their derivatives after oxidation [6][7][8][9] A D-luciferin hydrazine analogue has been synthesized for the selective imagi Cu 2+ . The CL reaction relies on the catalytic transformation of hydrazide analogue luciferin that is further converted to oxyluciferin by luciferase in the presence of ATP Mg 2+ (Scheme 3) [53]. A D-luciferin hydrazine analogue has been synthesized for the selective imaging of Cu 2+ . The CL reaction relies on the catalytic transformation of hydrazide analogue to D-luciferin that is further converted to oxyluciferin by luciferase in the presence of ATP and Mg 2+ (Scheme 3) [53]. A D-luciferin hydrazine analogue has been synthesized for the selective imaging of Cu 2+ . The CL reaction relies on the catalytic transformation of hydrazide analogue to Dluciferin that is further converted to oxyluciferin by luciferase in the presence of ATP and Mg 2+ (Scheme 3) [53]. Besides the commonly used luminol and cypridina luciferin scaffolds, other compounds such as peroxyoxalates, dioxetane, acridinium and chromone derivatives were also developed and have been used lately as efficient, direct CL reagents in various applications. A summary of recent direct CL applications is provided in Table 2 along with details on the system and the scaffold used. Besides the commonly used luminol and cypridina luciferin scaffolds, other compounds such as peroxyoxalates, dioxetane, acridinium and chromone derivatives were also developed and have been used lately as efficient, direct CL reagents in various applications. A summary of recent direct CL applications is provided in Table 2 along with details on the system and the scaffold used.

Direct Chemiluminescence by Luminol and Its Derivatives
In aqueous luminol solutions, H2O2 usually acts as an oxidant causing CL. However, luminol can also be oxidized catalytically by ozone, 1   It is also reported that luminol CL could be significantly enhanced in the presence of hydroxylated intermediates generated by persulfate-based advanced oxidation processes (AOPs) of 1,2-divinylbenzene (DVB). Since the emission of light found to be dependent on the oxidation efficiency of the recalcitrant pollutants, luminol CL was proposed ( Figure  4) for tracking a wider range of hydroxylated intermediates generated during persulfatebased AOPs under different degradation conditions, and other relevant organic compounds [55]. It is also reported that luminol CL could be significantly enhanced in the presence of hydroxylated intermediates generated by persulfate-based advanced oxidation processes (AOPs) of 1,2-divinylbenzene (DVB). Since the emission of light found to be dependent on the oxidation efficiency of the recalcitrant pollutants, luminol CL was proposed ( Figure 4) for tracking a wider range of hydroxylated intermediates generated during persulfate-based AOPs under different degradation conditions, and other relevant organic compounds [55].
In most cases, where transition metals are involved, metal-peroxide complexes and metal-luminol or metal-luminol-O 2 intermediates (M-LHOOH) are formed, which are believed to be the key intermediates for the CL of luminol-based systems (Scheme 5) [65].
Cobalt-doped hydroxyapatite nanoparticles (CoHA-NPs) have been reported to enhance luminol CL intensity by more than 75 times compared to the conventional luminol-H 2 O 2 system. During investigations on the impact of CoHA-NPs sizes, composition, and crystallinity on the CL intensity, it was found that the maximum CL intensity is achieved by polycrystalline NPs, owing to their larger surface area. The proposed mechanism involves the ionization of H 2 O 2 under basic conditions, leading to the formation of hydroperoxideanions, which subsequently interact with the Co atoms on the NPs surface, forming the metal-hydroperoxide intermediate. The peroxyl radicals, after decomposition of the intermediate, are degraded into HO • , which ultimately oxidizes luminol leading to the chemiexcited intermediate ( Figure 5) [56].  Cobalt-doped hydroxyapatite nanoparticles (CoHA-NPs) have been rep hance luminol CL intensity by more than 75 times compared to the convention H2O2 system. During investigations on the impact of CoHA-NPs sizes, comp crystallinity on the CL intensity, it was found that the maximum CL intensity by polycrystalline NPs, owing to their larger surface area. The proposed me volves the ionization of H2O2 under basic conditions, leading to the formation eroxide-anions, which subsequently interact with the Co atoms on the NPs su ing the metal-hydroperoxide intermediate. The peroxyl radicals, after decom the intermediate, are degraded into HO • , which ultimately oxidizes lumino the chemiexcited intermediate ( Figure 5) [56].  In most cases, where transition metals are involved, metal-pero metal-luminol or metal-luminol-O2 intermediates (M-LHOOH) are fo lieved to be the key intermediates for the CL of luminol-based system  In most cases, where transition metals are involved, metal-peroxide complexes and metal-luminol or metal-luminol-O2 intermediates (M-LHOOH) are formed, which are believed to be the key intermediates for the CL of luminol-based systems (Scheme 5) [65]. Cobalt-doped hydroxyapatite nanoparticles (CoHA-NPs) have been reported to enhance luminol CL intensity by more than 75 times compared to the conventional luminol-H2O2 system. During investigations on the impact of CoHA-NPs sizes, composition, and crystallinity on the CL intensity, it was found that the maximum CL intensity is achieved by polycrystalline NPs, owing to their larger surface area. The proposed mechanism involves the ionization of H2O2 under basic conditions, leading to the formation of hydroperoxide-anions, which subsequently interact with the Co atoms on the NPs surface, forming the metal-hydroperoxide intermediate. The peroxyl radicals, after decomposition of the intermediate, are degraded into HO • , which ultimately oxidizes luminol leading to the chemiexcited intermediate ( Figure 5) [56]. A new artificial heme-enzyme, so-called FeMC6*a [57], was also found to enhance luminol CL emission signal in comparison to the horseradish peroxidase (HRP)-based sensors ( Figure 6), when used in a H2O2-detecting bioassay [49,[66][67][68][69][70][71]. A new artificial heme-enzyme, so-called FeMC6*a [57], was also found to enhance luminol CL emission signal in comparison to the horseradish peroxidase (HRP)-based sensors ( Figure 6), when used in a H 2 O 2 -detecting bioassay [49,[66][67][68][69][70][71]. The catalytic activity of Cu-metal-organic frameworks (Cu-MOFs) with flower morphology, obtained from Cu-based metal-organic gels, was reported to promote persistent CL in luminol-H2O2 systems. According to the proposed mechanism, luminol's CL was attributed to the gradual generation of HO • , O2 • ‾ and 1 O2 species ( Figure 7) and recombination of HO • and O2 • ‾ into 1 O2 on the surface of Cu-MOFs, which also resulted in prolonged CL [58]. The catalytic activity of Cu-metal-organic frameworks (Cu-MOFs) with flower morphology, obtained from Cu-based metal-organic gels, was reported to promote persistent  The catalytic activity of Cu-metal-organic frameworks (Cu-MOFs) with flower morphology, obtained from Cu-based metal-organic gels, was reported to promote persistent CL in luminol-H2O2 systems. According to the proposed mechanism, luminol's CL was attributed to the gradual generation of HO • , O2 • ‾ and 1 O2 species ( Figure 7) and recombination of HO • and O2 • ‾ into 1 O2 on the surface of Cu-MOFs, which also resulted in prolonged CL [58]. Recently, it was also reported that luminol's CL efficiency can be tuned by manipulating the electronic properties of the aromatic ring via substitution, with the 8-methyl substituent providing the highest ΦCL enhancement ( Figure 8). In silico studies revealed that the 8-substitution assists the cleavage of the O-O bond via the negative charge donation to the in situ formed peroxide [72]. Previous studies on the factors that could possibly influence luminol's CL indicated that both steric and electronic effects play a significant role [73].

Direct Chemiluminescence from Probes Other Than Luminol
Apart from luminol derivatives, isoluminol [73], pyridopyridazines and phthalhydrazides [59] that were studied and used as CL probes [74], other molecules have been also designed and used in various CL applications which are not based on luminol's chemical structure. J. Yang and coworkers reported the synthesis and validation of a new CL molecule, so-called ADLumin-1, as a turn-on probe for amyloid beta (Aβ) species with maximum emission at 590 nm. This probe was then found that can be used in indirect CL via CRET to a curcumin-based NIR fluorescent imaging probe, so-called CRANAD-3, thus more information will be provided in the section on indirect CL (vide infra) [60].
Peroxyoxalate chemiluminescence (PO-CL) is one of three most common CL reactions [75]. It is a base-catalyzed reaction of an aromatic oxalic ester with H2O2 in the Recently, it was also reported that luminol's CL efficiency can be tuned by manipulating the electronic properties of the aromatic ring via substitution, with the 8-methyl substituent providing the highest Φ CL enhancement ( Figure 8). In silico studies revealed that the 8-substitution assists the cleavage of the O-O bond via the negative charge donation to the in situ formed peroxide [72]. Previous studies on the factors that could possibly influence luminol's CL indicated that both steric and electronic effects play a significant role [73]. The catalytic activity of Cu-metal-organic frameworks (Cu-MOFs) with flower morphology, obtained from Cu-based metal-organic gels, was reported to promote persistent CL in luminol-H2O2 systems. According to the proposed mechanism, luminol's CL was attributed to the gradual generation of HO • , O2 • ‾ and 1 O2 species (Figure 7) and recombination of HO • and O2 • ‾ into 1 O2 on the surface of Cu-MOFs, which also resulted in prolonged CL [58]. Recently, it was also reported that luminol's CL efficiency can be tuned by manipulating the electronic properties of the aromatic ring via substitution, with the 8-methyl substituent providing the highest ΦCL enhancement (Figure 8). In silico studies revealed that the 8-substitution assists the cleavage of the O-O bond via the negative charge donation to the in situ formed peroxide [72]. Previous studies on the factors that could possibly influence luminol's CL indicated that both steric and electronic effects play a significant role [73].

Direct Chemiluminescence from Probes Other Than Luminol
Apart from luminol derivatives, isoluminol [73], pyridopyridazines and phthalhydrazides [59] that were studied and used as CL probes [74], other molecules have been also designed and used in various CL applications which are not based on luminol's chemical structure. J. Yang and coworkers reported the synthesis and validation of a new CL molecule, so-called ADLumin-1, as a turn-on probe for amyloid beta (Aβ) species with maximum emission at 590 nm. This probe was then found that can be used in indirect CL via CRET to a curcumin-based NIR fluorescent imaging probe, so-called CRANAD-3, thus more information will be provided in the section on indirect CL (vide infra) [60].
Peroxyoxalate chemiluminescence (PO-CL) is one of three most common CL reactions [75]. It is a base-catalyzed reaction of an aromatic oxalic ester with H2O2 in the

Direct Chemiluminescence from Probes Other Than Luminol
Apart from luminol derivatives, isoluminol [73], pyridopyridazines and phthalhydrazides [59] that were studied and used as CL probes [74], other molecules have been also designed and used in various CL applications which are not based on luminol's chemical structure. J. Yang and coworkers reported the synthesis and validation of a new CL molecule, so-called ADLumin-1, as a turn-on probe for amyloid beta (Aβ) species with maximum emission at 590 nm. This probe was then found that can be used in indirect CL via CRET to a curcumin-based NIR fluorescent imaging probe, so-called CRANAD-3, thus more information will be provided in the section on indirect CL (vide infra) [60].
Peroxyoxalate chemiluminescence (PO-CL) is one of three most common CL reactions [75]. It is a base-catalyzed reaction of an aromatic oxalic ester with H 2 O 2 in the presence of a fluorophore, also commonly referred to as activator (ACT) [76]. When ACT interacts with the in situ-generated high-energy intermediate (HEI), usually a 1,2dioxetanedione, light emission occurs. The process relies on the reaction between the diphenyloxalate and H 2 O 2 that generates 1,2-dioxetanedione [77] which spontaneously decomposes to carbon dioxide via a paramagnetic oxalate biradical intermediate [78,79]. Several catalysts have been proposed so far, with imidazole being the most commonly used one [80]. Imidazole acts as a nucleophilic and base catalyst at different reaction steps [81]. Apart from imidazole, only few catalysts appear to work efficiently, and among them are: sodium salicylate, 4-N,N-dimethylaminopyridine and its derivatives, and a mixture of 1,2,4-triazole and 1,2,2,6,6-pentamethylpiperidine [82,83]. Cabello et al., working on the kinetics of the PO-CL, used three oxalic esters of diverse reactivity (e.g., bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl)oxalate (BMePO), bis[2-(methoxycarbonyl)-phenyl]oxalate (DMO)). The mechanistic studies performed in the presence of sodium salicylate in partially aqueous medium showed that the reaction rate constants were determined by H 2 O 2 and sodium salicylate amounts (Scheme 6). Further studies showed that by increasing the salicylate concentration there is a decrease in the CL efficiency, probably due to the interaction between salicylate and the in situ-produced HEI. From experiments using different oxalic esters, a variation in the CL emission was reported, with DMO showing the highest quantum yields in the aqueous medium used 1,2,4-triazole and 1,2,2,6,6-pentamethylpiperidine [82,83]. Cabello et al., wor kinetics of the PO-CL, used three oxalic esters of diverse reactivity (e.g., bis(2 rophenyl) oxalate (TCPO), bis(4-methylphenyl)oxalate (BMePO), bis[2-(m bonyl)-phenyl]oxalate (DMO)). The mechanistic studies performed in the pre dium salicylate in partially aqueous medium showed that the reaction rate con determined by H2O2 and sodium salicylate amounts (Scheme 6). Further stud that by increasing the salicylate concentration there is a decrease in the CL probably due to the interaction between salicylate and the in situ-produced experiments using different oxalic esters, a variation in the CL emission wa with DMO showing the highest quantum yields in the aqueous medium use DME/H2O) [84]. Scheme 6. Mechanism of PO-CL between an oxalic ester and H2O2, occurring via spe talysis (SBS) by sodium salicylate, through the formation of (a) hydroperoxy anion, (b) alic ester and (c) 1,2-dioxetanedione, which subsequently leads to (d) carbon dioxide an sion.
When 2,6-lutidine was used as a non-nucleophilic base catalyst on the T action, it was found that the concentrations of 2,6-lutidine and H2O2 play a cr the rate-determining step. The reaction was found to follow a general base cata anism (Scheme 7) [85]. Scheme 7. Reaction mechanism of TCPO with H2O2 in DME under general base cat lutidine (Lut) towards CL emission in the presence of a fluorophore. When 2,6-lutidine was used as a non-nucleophilic base catalyst on the TCPO CL reaction, it was found that the concentrations of 2,6-lutidine and H 2 O 2 play a crucial role in the rate-determining step. The reaction was found to follow a general base catalysis mechanism (Scheme 7) [85]. rophenyl) oxalate (TCPO), bis(4-methylphenyl)oxalate (BMePO), bis[2-(meth bonyl)-phenyl]oxalate (DMO)). The mechanistic studies performed in the presen dium salicylate in partially aqueous medium showed that the reaction rate constan determined by H2O2 and sodium salicylate amounts (Scheme 6). Further studies that by increasing the salicylate concentration there is a decrease in the CL ef probably due to the interaction between salicylate and the in situ-produced HE experiments using different oxalic esters, a variation in the CL emission was r with DMO showing the highest quantum yields in the aqueous medium used (1 DME/H2O) [84]. Scheme 6. Mechanism of PO-CL between an oxalic ester and H2O2, occurring via specific talysis (SBS) by sodium salicylate, through the formation of (a) hydroperoxy anion, (b) mo alic ester and (c) 1,2-dioxetanedione, which subsequently leads to (d) carbon dioxide and li sion.
When 2,6-lutidine was used as a non-nucleophilic base catalyst on the TCPO action, it was found that the concentrations of 2,6-lutidine and H2O2 play a crucia the rate-determining step. The reaction was found to follow a general base catalys anism (Scheme 7) [85]. Scheme 7. Reaction mechanism of TCPO with H2O2 in DME under general base catalys lutidine (Lut) towards CL emission in the presence of a fluorophore. Among other CL molecules of interest are the 1,10-phenanthroline dioxetanes, generated after treatment of 1,10-phenanthroline with IO 4 − in the presence of H 2 O 2 . It has been reported that 1,10-phenanthroline dioxetanes enhance light emission in respect to the IO 4 − -H 2 O 2 system alone. The proposed radical-based mechanism involves the C 5 =C 6 bond of 1,10-phenanthroline that is initially attacked by HO • , leading to a dioxetane intermediate responsible for the CL emission (Scheme 8) [86]. The CL of the IO 4 − /peroxide system and the factors influencing the emission have been recently reviewed elsewhere [87].
Among other CL molecules of interest are the 1,10-phenanthroline dioxetanes, generated after treatment of 1,10-phenanthroline with IO4 − in the presence of H2O2. It has been reported that 1,10-phenanthroline dioxetanes enhance light emission in respect to the IO4 − -H2O2 system alone. The proposed radical-based mechanism involves the C 5 =C 6 bond of 1,10-phenanthroline that is initially attacked by HO • , leading to a dioxetane intermediate responsible for the CL emission (Scheme 8) [86]. The CL of the IO4 − /peroxide system and the factors influencing the emission have been recently reviewed elsewhere [87]. Investigation into the CL efficiency of four bicyclic dioxetanes bearing 2-hydroxybiphenyl-4-yl (a), 2-hydroxy-p-terphenyl-4-yl (b), 2-hydroxy-p-quaterphenyl-4-yl (c), or 2hydroxy-p-quinquephenyl-4-yl (d) (Figure 9) moieties, showed that a-c emit light from 466 nm to 547 nm during their decomposition while d was found to emit light of much weaker intensity from 490 nm up to 607 nm, depending on the solvent (water, acetonitrile or DMSO). The CL efficiencies of the latter dioxetanes in aqueous media were found to be enhanced in the presence of additives such as β-MCD (β-methylated cyclodextrin) or TBHP (tributylhexadecylphosphonium bromide) [88]. the factors influencing the emission have been recently reviewed elsewhere [87]. Investigation into the CL efficiency of four bicyclic dioxetanes bearing 2-hydroxybiphenyl-4-yl (a), 2-hydroxy-p-terphenyl-4-yl (b), 2-hydroxy-p-quaterphenyl-4-yl (c), or 2hydroxy-p-quinquephenyl-4-yl (d) (Figure 9) moieties, showed that a-c emit light from 466 nm to 547 nm during their decomposition while d was found to emit light of much weaker intensity from 490 nm up to 607 nm, depending on the solvent (water, acetonitrile or DMSO). The CL efficiencies of the latter dioxetanes in aqueous media were found to be enhanced in the presence of additives such as β-MCD (β-methylated cyclodextrin) or TBHP (tributylhexadecylphosphonium bromide) [88].  In the presence of 1 O 2 , the probe is oxidized, affording the phenol-dioxetane moiety which undergoes spontaneous bond cleavage leading to the formation of the chemiexcited product responsible for the CL emission ( Figure 10). With this probe, they were able to detect 1 O 2 in vitro with a turn-on bathochromic CL signal in the NIR region at 700 nm and image intracellular 1 O 2 produced by a photosensitizer under simulated photodynamic therapy conditions [54]. Similarly, hyperconjugated push-pull dicyanomethylchromone systems were used for the in vivo detection of H 2 O 2 and β-galactosidase activity [61]. ety which undergoes spontaneous bond cleavage leading to the formation of the chemiexcited product responsible for the CL emission ( Figure 10). With this probe, they were able to detect 1 O2 in vitro with a turn-on bathochromic CL signal in the NIR region at 700 nm and image intracellular 1 O2 produced by a photosensitizer under simulated photodynamic therapy conditions [54]. Similarly, hyperconjugated push-pull dicyanomethylchromone systems were used for the in vivo detection of H2O2 and β-galactosidase activity [61]. The CL emission mechanism proposed for the Schaap's dioxetane probes [89][90][91], involves Chemically Initiated Electron Exchange Luminescence (CIEEL) [1,92] with a solvent-cage back electron-transfer step, which affords the 2-adamantanone and the chemiexcited phenolate that is responsible for the light emission (Scheme 9) [92,93]. The CL emission mechanism proposed for the Schaap's dioxetane probes [89][90][91], involves Chemically Initiated Electron Exchange Luminescence (CIEEL) [1,92] with a solventcage back electron-transfer step, which affords the 2-adamantanone and the chemiexcited phenolate that is responsible for the light emission (Scheme 9) [92,93]. ety which undergoes spontaneous bond cleavage leading to the formation of the chemiexcited product responsible for the CL emission ( Figure 10). With this probe, they were able to detect 1 O2 in vitro with a turn-on bathochromic CL signal in the NIR region at 700 nm and image intracellular 1 O2 produced by a photosensitizer under simulated photodynamic therapy conditions [54]. Similarly, hyperconjugated push-pull dicyanomethylchromone systems were used for the in vivo detection of H2O2 and β-galactosidase activity [61]. The CL emission mechanism proposed for the Schaap's dioxetane probes [89][90][91], involves Chemically Initiated Electron Exchange Luminescence (CIEEL) [1,92] with a solvent-cage back electron-transfer step, which affords the 2-adamantanone and the chemiexcited phenolate that is responsible for the light emission (Scheme 9) [92,93].  Shabat and coworkers described the development of functional self-immolative mo lecular scaffolds based on Schaap's dioxetane probes, and described their use in sign amplification and real-time monitoring of enzymatic activity or drug release. The gener mode of action involves a triggering event for releasing a chemiexcited product, the C reporter unit, and the payload which can be a reagent molecule, a drug or a prodrug. Th strategy was used successfully in monitored release of the cytotoxic agent monometh auristatin E (MMAE) from prodrug Gal-A-Pept (Scheme 11). The prodrug is initially re ognized by a β-galactosidase, releasing intermediate A-Pept which undergoes 1,6-elim nation to afford the MMAE drug and quinone-methide A. Then, intermediate B is pro duced by the reaction of A with H2O, subsequently releasing 2-adamantanone and th chemiexcited phenolate C*. Green emission at 550 nm is observed due to radiative deca of the phenolate to the ground state [62]. Shabat and coworkers described the development of functional self-immolative molecular scaffolds based on Schaap's dioxetane probes, and described their use in signal amplification and real-time monitoring of enzymatic activity or drug release. The general mode of action involves a triggering event for releasing a chemiexcited product, the CL reporter unit, and the payload which can be a reagent molecule, a drug or a prodrug. This strategy was used successfully in monitored release of the cytotoxic agent monomethyl auristatin E (MMAE) from prodrug Gal-A-Pept (Scheme 11). The prodrug is initially recognized by a β-galactosidase, releasing intermediate A-Pept which undergoes 1,6-elimination to afford the MMAE drug and quinone-methide A. Then, intermediate B is produced by the reaction of A with H 2 O, subsequently releasing 2-adamantanone and the chemiexcited phenolate C*. Green emission at 550 nm is observed due to radiative decay of the phenolate to the ground state [62]. Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatments due to the acquisition of antibiotic resistance. Recently, Das et al. demonstrated the first carbapenemase CL probe (CPCL) for the detection of carbapenemase and CPOs. The unique structural design of CPCL enables the probe to rapidly disassemble via an efficient 1,8-elimination process upon hydrolysis of the β-lactam ring by a carbapenemase (Figure 11). This process results in the release of a chemiexcited phenolate that undergoes radiative relaxation, producing photons at 540 nm that enable a rapid and selective detection of carbapenemase activity [63]. Scheme 11. Activation mode of the self-immolative chemiluminescence prodrug Gal-A-Pept by βgalactosidase.
Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatments due to the acquisition of antibiotic resistance. Recently, Das et al. demonstrated the first carbapenemase CL probe (CPCL) for the detection of carbapenemase and CPOs. The unique structural design of CPCL enables the probe to rapidly disassemble via an efficient 1,8-elimination process upon hydrolysis of the β-lactam ring by a carbapenemase (Figure 11). This process results in the release of a chemiexcited phenolate that undergoes radiative relaxation, producing photons at 540 nm that enable a rapid and selective detection of carbapenemase activity [63]. The same as above reporter for monitoring carbapenemase activity CL was also used for detection of prostate-specific antigen (PSA) enzymatic activity by using artificial metalloenzymes (ArMs). The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane intermediate affording a chemiexcited benzoate that emits a green photon. The benefit and simplicity The same as above reporter for monitoring carbapenemase activity CL was also used for detection of prostate-specific antigen (PSA) enzymatic activity by using artificial metalloenzymes (ArMs). The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane intermediate affording a chemiexcited benzoate that emits a green photon. The benefit and simplicity of a CL assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer [64].
Other efficient CL molecules are based on acridinium ester dyes analogues. The acridan dyes are quaternary acridine derivatives that become luminescent directly after their activation [95][96][97][98][99][100]. The mechanism of CL involving a dioxetane intermediate was first introduced by McCapra (Scheme 12) [101,102]. The simplicity of their synthesis and the high quantum yield (up to ca. 7%) [102], together with their ability to become oxidized under alkaline conditions, making acridinium derivatives highly suitable for sensing and chemiluminometric analysis [95,103]. Research on the design of new acridinium derivatives with even higher activity is an ongoing process [104].
Acridine-9-carboxylate derivatives [e.g., (4-(methoxycarbonyl)phenyl-10-methyl-10λ 4 -2,7-dimethyl-acridine-9-carboxylates)] were used for studying the CL efficiency in the 7-10 pH range. Reactions of acridine-9-carboxylates with H 2 O 2 result in the formation of a relatively unstable dioxetanone intermediates. The chemiexcited methyl-acridone products formed after the decomposition of the dioxetanone intermediates are responsible for light emission. Among the compounds studied, 4-(methoxycarbonyl)phenyl-derivative gave the highest CL intensities at pH 7−10, indicating that the introduction of electron-donating groups at the 2,7-positions on the acridine moiety plays a crucial role in the whole process (Scheme 13) [105]. activation [95][96][97][98][99][100]. The mechanism of CL involving a dioxetane intermediate was first introduced by McCapra (Scheme 12) [101,102]. The simplicity of their synthesis and the high quantum yield (up to ca. 7%) [102], together with their ability to become oxidized under alkaline conditions, making acridinium derivatives highly suitable for sensing and chemiluminometric analysis [95,103]. Research on the design of new acridinium derivatives with even higher activity is an ongoing process [104].

Indirect Chemiluminescence
In the indirect CL process, energy is transferred by the chemiexcited mole ergy donor, D) to a second molecule (acceptor, E), which is further excited d process. In the resonance excitation transfer mechanism, D and E are not in co may be separated by as much as 10 nm, although 1 nm or smaller transfer dist more convenient. Except for distance, other factors dictating the energy transfe the lifetime of the excited state of D and the degree of the overlap between the spectrum of D and the absorption spectrum of E. Usually, the higher overlapping efficient the energy transfer from D to E. The solvent is another crucial factor fo ergy transfer between D and E molecules, however this effect includes a lot o characteristics properties of each system, such as the solvent cage factor. The sh energy transfer by the wavefunction overlap between the donor and the accept ing overlap between the electronic clouds, is also a crucial factor. Particularly, Förster Resonance Energy Transfer or Fluorescence Resonance Energy Transfe Scheme 13. Proposed CL mechanism of 4-(methoxycarbonyl)phenyl-10-methyl-10λ 4 -2,7-dimethylacridine-9-carboxylates.

Indirect Chemiluminescence
In the indirect CL process, energy is transferred by the chemiexcited molecule (energy donor, D) to a second molecule (acceptor, E), which is further excited during the process. In the resonance excitation transfer mechanism, D and E are not in contact and may be separated by as much as 10 nm, although 1 nm or smaller transfer distances are more convenient. Except for distance, other factors dictating the energy transfer include the lifetime of the excited state of D and the degree of the overlap between the emission spectrum of D and the absorption spectrum of E. Usually, the higher overlapping the more efficient the energy transfer from D to E. The solvent is another crucial factor for the energy transfer between D and E molecules, however this effect includes a lot of specific characteristics properties of each system, such as the solvent cage factor. The shortrange energy transfer by the wavefunction overlap between the donor and the acceptor, meaning overlap between the electronic clouds, is also a crucial factor. Particularly, both the Förster Resonance Energy Transfer or Fluorescence Resonance Energy Transfer (FRET) and Dexter Energy Transfer, with the second occurring when the D-E wavefunctions overlap, contribute to the energy transfer of the system, promoting its scope. FRET, which refers to the nonradiative energy transfer from an excited donor to an acceptor mediated by electronic dipole-dipole coupling, between a CL reagent as the excited donor and a fluorophore acceptor, is referred to as chemiluminescence resonance energy transfer (CRET) [106].
Two different modes of indirect CL emission can be distinguished, intermolecular and intramolecular CL, as shown in a simplified manner in Scheme 14A,B, respectively. After the triggering event, chemiexcited D is formed and then its energy is transferred to E, e.g., a photosensitizer (PS). When E is not directly linked with the chemiexcited species (Scheme 14A), the intermolecular distance between D and E (photosensitizer) plays crucial role in the energy transfer, with a direct emission ("escaping" photons) taking place together with the secondary emission. Sometimes this mode of action is crucial for distinguishing the two different events and their efficacy rate: one of the initial CL emission (direct emission) and the second is processed from the excited acceptor properties (Scheme 14A, secondary emission). On the other hand, in the case of an intramolecular system, the chemiexcited donor (D) is linked with the energy acceptor molecule (E) and a secondary emission pathway is favored (Scheme 14B). The absorbance band of conventional photosensitizers such as Rose Bengal and TetraPhenylPorphyrin (TPP) (Type II photosensitizers) [107][108][109][110], as well as W10O32 4-(Type I' photocatalyst) [111][112][113], fit well into the requirements for efficient CRET and high CL emissions [114]. Quantum Dots (QDs) have been also used successfully in various CRET studies. QDs are luminescent semiconductor nanocrystals with very distinct, sizedependent physicochemical behavior [106]. It should be noted that the excited photosensitizer or QDs can trigger various reaction cascades. For example, in the presence of molecular oxygen they can result in the production of ROS (e.g., 1 O2, HO • , O2 •− ). This function is used in CL-induced photodynamic therapies and is described later in the text in more details (vide infra) [115].
A summary of recent direct CL scaffolds, systems and their applications are provided in Table 3 and Table 4, based on intra-and inter-molecular modes of reaction.  [111][112][113], fit well into the requirements for efficient CRET and high CL emissions [114]. Quantum Dots (QDs) have been also used successfully in various CRET studies. QDs are luminescent semiconductor nanocrystals with very distinct, size-dependent physicochemical behavior [106]. It should be noted that the excited photosensitizer or QDs can trigger various reaction cascades. For example, in the presence of molecular oxygen they can result in the production of ROS (e.g., 1 O 2 , HO • , O 2 •− ). This function is used in CL-induced photodynamic therapies and is described later in the text in more details (vide infra) [115].
A summary of recent direct CL scaffolds, systems and their applications are provided in Tables 3 and 4, based on intra-and inter-molecular modes of reaction.

Intermolecular Chemiluminescence Emission Mode
J. Yang and coworkers, as mentioned above (vide supra), reported on the ADLumin-1 probe for Aβ species (Figure 12). To overcome problems related to short emission of ADLumin-1 they used the CRET strategy for transferring the energy to CRANAD-3, a curcumin-based, NIR fluorescent imaging probe emitting in the near-infrared region, at 730 nm (vide supra) [60].
Molecules 2021, 26,7664 carbonate moiety triggered the self-immolative cleavage, affording the che nolate. Then, the energy transfer via CRET to the Emerald II TM CL enhance detection at 545 nm ( Figure 14) [122]. Another important study by Lipper et al., described that CL energy Schaap's dioxetane probe can be efficiently used for ratiometric pH ima proach was based on energy transfer after the emission of phenolate durin laxation to the pH-sensitive dye carboxy-SNARF-1 in CL-enhancer soluti thee relative intensities at 535 nm, 585 nm and 650 nm due to relaxation of and deprotonated dye, respectively ( Figure 15) [116]. Another important study by Lipper et al., described that CL energy transfer from Schaap's dioxetane probe can be efficiently used for ratiometric pH imaging. Their approach was based on energy transfer after the emission of phenolate during radiative relaxation to the pH-sensitive dye carboxy-SNARF-1 in CL-enhancer solutions, providing thee relative intensities at 535 nm, 585 nm and 650 nm due to relaxation of the protonated and deprotonated dye, respectively ( Figure 15) [116].
Recently, a supramolecular strategy was reported, achieving a highly efficient CLinitiated cascade FRET by following a three-criteria rule: (i) D emission and the E absorption spectral overlapping; (ii) 10 nm or less as the D-E distance; and (iii) orientation of the D and E dipoles (see also Scheme 14). For this purpose, a nanoassemble β-cyclodextrin (β-CD) was used in an aqueous luminol/H 2 O 2 solution in the presence of various fluorophores, which enable an accelerated multicolor CL with flexible emission wavelength in the range of 410-610 nm. Hydroxypropyl methylcellulose was utilized to slow down the diffusion rate, while the reaction was buffered by addition of solid Ca(OH) 2 . Moreover, it was found that Co 2+ -chitosan increased the CL emission of the system when merged with the β-CD nanoassembly system [126]. Another important study by Lipper et al., described that CL energy transfer from Schaap's dioxetane probe can be efficiently used for ratiometric pH imaging. Their approach was based on energy transfer after the emission of phenolate during radiative relaxation to the pH-sensitive dye carboxy-SNARF-1 in CL-enhancer solutions, providing thee relative intensities at 535 nm, 585 nm and 650 nm due to relaxation of the protonated and deprotonated dye, respectively ( Figure 15) [116]. Recently, a supramolecular strategy was reported, achieving a highly efficient CLinitiated cascade FRET by following a three-criteria rule: (i) D emission and the E absorption spectral overlapping; (ii) 10 nm or less as the D-E distance; and (iii) orientation of the D and E dipoles (see also Scheme 14). For this purpose, a nanoassemble β-cyclodextrin (β-CD) was used in an aqueous luminol/H2O2 solution in the presence of various fluorophores, which enable an accelerated multicolor CL with flexible emission wavelength in the range of 410-610 nm. Hydroxypropyl methylcellulose was utilized to slow down the diffusion rate, while the reaction was buffered by addition of solid Ca(OH)2. Moreover, it Another interesting CL system of cyclic peroxides was catalytically produced by tetrahydrofuran hydrogen peroxide in the presence of BSA-stabilized Gold Nanoclusters (Au@BSA NCs). Gold nanoclusters were found to accelerate the decomposition of the THF-hydroperoxides and promoted CRET between the chemiexcited dioxetane derivatives and the Au@BSA NCs ( Figure 16). The CL emission (λ max = 650 nm) was found to be further enhanced by the presence of copper ions [127].
Molecules 2021, 26, 7664 19 of 30 was found that Co 2+ -chitosan increased the CL emission of the system when merged with the β-CD nanoassembly system [126]. Another interesting CL system of cyclic peroxides was catalytically produced by tetrahydrofuran hydrogen peroxide in the presence of BSA-stabilized Gold Nanoclusters (Au@BSA NCs). Gold nanoclusters were found to accelerate the decomposition of the THF-hydroperoxides and promoted CRET between the chemiexcited dioxetane derivatives and the Au@BSA NCs ( Figure 16). The CL emission (λmax = 650 nm) was found to be further enhanced by the presence of copper ions [127]. From the investigation into the CL behavior of an amphiphilic conjugate (defined as CLP) NPs, it was revealed that the NPs consisting of chlorine6 (Ce6), conjugated with luminol and polyethylene glycol undergo CRET with energy transfer to Ce6 upon triggering with H2O2. The CLP NPs were tested in cancer cells with high expression of H2O2 and monitored at 660-740 nm. In addition, excited Ce6 was capable of producing 1 O2 in situ PDT against H2O2-high tumors, inhibiting lung metastasis. In vitro and in vivo experiments showed that CLP NPs could be a promising system in nanotheranostics, providing imaging and therapy, simultaneously, particularly in tumors with high levels of H2O2 [128].
Black Phosphorene (BP) QDs were initially studied in CL experiments upon reaction with H2O2 and HClO by Y. Lv and coworkers [129]. The same group two years later reported on the extension of the study with NH2-functionalized BP QDs (N-BPQDs) and showed that surface modification dictates the energy gap and electron mobility, thus favoring CL emission at 500 nm after triggering by persulfates [130].
Luminol's analogue, L012, that shows 100 times higher than luminol CL intensity after oxidation by H2O2 at pH 7, was also used as a CRET donor. In vitro and in vivo studies From the investigation into the CL behavior of an amphiphilic conjugate (defined as CLP) NPs, it was revealed that the NPs consisting of chlorine6 (Ce6), conjugated with luminol and polyethylene glycol undergo CRET with energy transfer to Ce6 upon triggering with H 2 O 2 . The CLP NPs were tested in cancer cells with high expression of H 2 O 2 and monitored at 660-740 nm. In addition, excited Ce6 was capable of producing 1 O 2 in situ PDT against H 2 O 2 -high tumors, inhibiting lung metastasis. In vitro and in vivo experiments showed that CLP NPs could be a promising system in nanotheranostics, providing imaging and therapy, simultaneously, particularly in tumors with high levels of H 2 O 2 [128].
Black Phosphorene (BP) QDs were initially studied in CL experiments upon reaction with H 2 O 2 and HClO by Y. Lv and coworkers [129]. The same group two years later reported on the extension of the study with NH 2 -functionalized BP QDs (N-BPQDs) and showed that surface modification dictates the energy gap and electron mobility, thus favoring CL emission at 500 nm after triggering by persulfates [130].
Luminol's analogue, L012, that shows 100 times higher than luminol CL intensity after oxidation by H 2 O 2 at pH 7, was also used as a CRET donor. In vitro and in vivo studies with polyenthylene(glycol)-coated CdSeTe core QDs, used as energy acceptors, demonstrated that they can image H 2 O 2 in live cells, pointing out the high potential of the system in the diagnosis of H 2 O 2 -associated diseases. The chemiexcited QDs were able to emit light at 780 nm, thus allowing monitoring [117].
The same luminol analogue L012 and H 2 O 2 were recently studied in the presence of Fe(III) deuteroporphyrin IX chloride-polymer dots (FeDP-Pdots) as CL catalysts towards cancer therapy, providing high concentrations of ROS due to high CRET efficiency ( Figure 17) [118].
showed that CLP NPs could be a promising system in nanotheranostics, providing ing and therapy, simultaneously, particularly in tumors with high levels of H2O2 [12 Black Phosphorene (BP) QDs were initially studied in CL experiments upon rea with H2O2 and HClO by Y. Lv and coworkers [129]. The same group two years lat ported on the extension of the study with NH2-functionalized BP QDs (N-BPQDs showed that surface modification dictates the energy gap and electron mobility, th voring CL emission at 500 nm after triggering by persulfates [130]. Luminol's analogue, L012, that shows 100 times higher than luminol CL inte after oxidation by H2O2 at pH 7, was also used as a CRET donor. In vitro and in vivo st with polyenthylene(glycol)-coated CdSeTe core QDs, used as energy acceptors, de strated that they can image H2O2 in live cells, pointing out the high potential of the sy in the diagnosis of H2O2-associated diseases. The chemiexcited QDs were able to emi at 780 nm, thus allowing monitoring [117].
The same luminol analogue L012 and H2O2 were recently studied in the presen Fe(III) deuteroporphyrin IX chloride-polymer dots (FeDP-Pdots) as CL catalysts tow cancer therapy, providing high concentrations of ROS due to high CRET efficiency (F 17) [118]. CL has been also used in polymerization reactions. Linear polymer chains were synthesized along with cross-linked functional polymeric networks as a result of CL photoinitiated cationic polymerization based on a sulfonium salt ( Figure 18). The polymerization reaction initiated CL emission at 430 nm. The photoinitiator decomposition after excitation produced protonic acids that were capable of initiating cationic polymerization of oxirane (epoxides) and vinyl monomers. The presence of iodonium salt in the initiating system was found to increase the polymerization efficiency [119]. CL has been also used in polymerization reactions. Linear polymer chains were synthesized along with cross-linked functional polymeric networks as a result of CL photoinitiated cationic polymerization based on a sulfonium salt ( Figure 18). The polymerization reaction initiated CL emission at 430 nm. The photoinitiator decomposition after excitation produced protonic acids that were capable of initiating cationic polymerization of oxirane (epoxides) and vinyl monomers. The presence of iodonium salt in the initiating system was found to increase the polymerization efficiency. [119]

Intramolecular Chemiluminescence Emission Mode
An intramolecular CRET approach based on chemically initiated electron exchange luminescence (CIEEL) has been recently adapted, while developing the first single molecule ratiometric CL probe for imaging pH in live animals. The proposed direct-indirect emission hybrid methodology, with emissions at 530 nm and 580 nm, relies on an acrylamide 1,2-dioxetane scaffold linked via a piperazine linker to a pH-sensitive carbofluorescein. This methodology withholds many characteristics that make it suitable for applications in quantifications of other important analytes produced in vivo ( Figure 19) [123].

Intramolecular Chemiluminescence Emission Mode
An intramolecular CRET approach based on chemically initiated electron exchange luminescence (CIEEL) has been recently adapted, while developing the first single molecule ratiometric CL probe for imaging pH in live animals. The proposed direct-indirect emission hybrid methodology, with emissions at 530 nm and 580 nm, relies on an acrylamide 1,2dioxetane scaffold linked via a piperazine linker to a pH-sensitive carbofluorescein. This methodology withholds many characteristics that make it suitable for applications in quantifications of other important analytes produced in vivo ( Figure 19) [123].
An intramolecular CRET approach based on chemically initiated electron exchange luminescence (CIEEL) has been recently adapted, while developing the first single molecule ratiometric CL probe for imaging pH in live animals. The proposed direct-indirect emission hybrid methodology, with emissions at 530 nm and 580 nm, relies on an acrylamide 1,2-dioxetane scaffold linked via a piperazine linker to a pH-sensitive carbofluorescein. This methodology withholds many characteristics that make it suitable for applications in quantifications of other important analytes produced in vivo ( Figure 19) [123]. Teranishi proposed a cypridina luciferin analogue for detecting O2 •-via intramolecular CRET to sulforhodamine 101. The energy transfer from the oxidized imidazopyrazinone to the acceptor dye results in light emission at 610 nm (Scheme 15). A similar approach was studied earlier with fluorescein analogue FCLA, emitting at 532 nm [124]. Teranishi proposed a cypridina luciferin analogue for detecting O 2 •− via intramolecular CRET to sulforhodamine 101. The energy transfer from the oxidized imidazopyrazinone to the acceptor dye results in light emission at 610 nm (Scheme 15). A similar approach was studied earlier with fluorescein analogue FCLA, emitting at 532 nm [124]. Α novel on-off CRET system based on phenoxy-dioxetane 7-hydroxycoumarin scaffold for light emission, upon slow chemiexcitation in aqueous solution, was recently reported. The energy donor is covalently linked to a quencher via a peptide linker that is recognizable by metalloproteinases. In the absence of enzymatic activity CRET is on, thus the energy is transferred to the quencher. When the peptide linker is cleaved by enzymes CRET is off, thus light emission is observed (Figure 20). The system was tested in cancer cells, showing good detection levels in matrix metalloproteinase activity [125]. A novel on-off CRET system based on phenoxy-dioxetane 7-hydroxycoumarin scaffold for light emission, upon slow chemiexcitation in aqueous solution, was recently reported. The energy donor is covalently linked to a quencher via a peptide linker that is recognizable by metalloproteinases. In the absence of enzymatic activity CRET is on, thus the energy is transferred to the quencher. When the peptide linker is cleaved by enzymes CRET is off, thus light emission is observed (Figure 20). The system was tested in cancer cells, showing good detection levels in matrix metalloproteinase activity [125].
fold for light emission, upon slow chemiexcitation in aqueous solution, was recently reported. The energy donor is covalently linked to a quencher via a peptide linker that is recognizable by metalloproteinases. In the absence of enzymatic activity CRET is on, thus the energy is transferred to the quencher. When the peptide linker is cleaved by enzymes CRET is off, thus light emission is observed (Figure 20). The system was tested in cancer cells, showing good detection levels in matrix metalloproteinase activity [125].

The Dual Role of Singlet Oxygen in Chemiluminescence
Singlet oxygen was discovered and confirmed spectroscopically in the early 1960s when the H 2 O 2 /hypochlorite system was at the epicenter of the CL studies [131][132][133][134][135][136][137][138]. Its emission at 1270 nm was attributed to the monomeric singlet oxygen and the emissions at 633 nm and 703 nm, to its dimeric aggregate form (Scheme 16). It is noteworthy that the energy difference between the ground state of O 2 ( 3 Σ g ) and its singlet excited state ( 1 ∆ g , singlet oxygen, 1 O 2 ) is 22.5 kcal/mol. Moreover, its lifetime depends significantly on the solvent (e.g.,~59 ms in carbon tetrachloride, 9.8 ± 0.6 µs in methanol-h 4 , 77 ± 4 µs acetonitrile and 3.7 ± 0.4 µs in water) [139,140]. During recent decades, an increasing number of studies have focused on exploring the role of 1 O2 in chemistry, biology, medicine and environmental and material sciences. Lately, the increasing interest of the scientific community in novel therapies has led to further exploration of 1 O2 against persisting diseases (i.e., cancer). Thus, a significant amount of the new studies are now focused on the generation modes and the photophysical properties [141], dictating its interaction with other biomolecules in live cells. On the other hand, 1 O2 is a very important oxidizing agent in organic chemistry transformations. There are two main approaches for generating 1 O2. The first, and more famous one, involves light and a photosensitizer, while the less famous one is hydroperoxide-based and it is often called the "in dark" process, since 1 O2 is produced in the absence of light. To avoid confusion between the CL-induced 1 O2production and the CL of the 1 O2 itself, we should distinguish here the two completely different CL. The energy transfer and radiative processes of the CL energy acceptors or chemiexcited intermediates are illustrated in Figure 21. During recent decades, an increasing number of studies have focused on exploring the role of 1 O 2 in chemistry, biology, medicine and environmental and material sciences. Lately, the increasing interest of the scientific community in novel therapies has led to further exploration of 1 O 2 against persisting diseases (i.e., cancer). Thus, a significant amount of the new studies are now focused on the generation modes and the photophysical properties [141], dictating its interaction with other biomolecules in live cells. On the other hand, 1 O 2 is a very important oxidizing agent in organic chemistry transformations. There are two main approaches for generating 1 O 2 . The first, and more famous one, involves light and a photosensitizer, while the less famous one is hydroperoxide-based and it is often called the "in dark" process, since 1 O 2 is produced in the absence of light. To avoid confusion between the CL-induced 1 O 2 production and the CL of the 1 O 2 itself, we should distinguish here the two completely different CL. The energy transfer and radiative processes of the CL energy acceptors or chemiexcited intermediates are illustrated in Figure 21.
The majority of the recent photochemical or CL studies on 1 O 2 applications employ well-known and established methodologies for generating an efficient amount of the oxidizing agent based on photosensitizers such as TTP, Rose Bengal, Cu 2+ [142], Ru 2+ and Ir 3+ complexes, phthalocyanines [143,144] and other analogues. Kellett and his coworkers have presented a class of di-copper (II) complexes based on the synthetic chemical nuclease [Cu(Phen) 2 ] + (where Phen = 1,10-phenathroline), that were selective against solid epithelial cancer cells from line panel NCI-60. Among other ROS, their agents are found to catalyze intracellular 1 O 2 contributing to oxidative DNA damage [145]. Recently, our team has also explored the role of Cu + complexes in the photochemical production of 1 O 2 . We found and presented for the first time that [Cu(Xantphos)(neoc)]BF 4 can be used as an alternative and very efficient photosensitizer in 1 O 2 -mediated C-H allylic oxygenations [146].
There are two main approaches for generating 1 O2. The first, and more famous one, involves light and a photosensitizer, while the less famous one is hydroperoxide-based and it is often called the "in dark" process, since 1 O2 is produced in the absence of light. To avoid confusion between the CL-induced 1 O2production and the CL of the 1 O2 itself, we should distinguish here the two completely different CL. The energy transfer and radiative processes of the CL energy acceptors or chemiexcited intermediates are illustrated in Figure 21. The majority of the recent photochemical or CL studies on 1 O2 applications employ well-known and established methodologies for generating an efficient amount of the oxidizing agent based on photosensitizers such as TTP, Rose Bengal, Cu 2+ [142], Ru 2+ and Ir 3+ complexes, phthalocyanines [143,144] and other analogues. Kellett and his coworkers have presented a class of di-copper (II) complexes based on the synthetic chemical nuclease [Cu(Phen)2] + (where Phen = 1,10-phenathroline), that were selective against solid epithelial cancer cells from line panel NCI-60. Among other ROS, their agents are found to catalyze intracellular 1 O2 contributing to oxidative DNA damage [145]. Recently, our team has also explored the role of Cu + complexes in the photochemical production of 1 O2. We found and presented for the first time that [Cu(Xantphos)(neoc)]BF4 can be used as an alternative and very efficient photosensitizer in 1 O2-mediated C-H allylic oxygenations [146].
Recently, in our group, the plausible production of 1 O2 was studied under different CL reaction conditions and in the presence of various sensitizers. In this study, except for Recently, in our group, the plausible production of 1 O 2 was studied under different CL reaction conditions and in the presence of various sensitizers. In this study, except for the common solvents used in CL procedures, diethylphthalates, the greener ethyl acetate was found to give unexpectedly good results. In addition, several photosensitizers, i.e., Rose Bengal (RB), Methylene Blue, 9,10-diphenylanthracene (9,10-DPA), Eosin Y and Rhodamine B, were tested for light emission through the CL process, with RB providing the best results based on the light emission duration. In the presence of TCPO, H 2 O 2 , RB, sodium salicylate and a mixture of ethyl acetate/acetonitrile in a ratio of 2/1, light emission was observed for 40-50 min (Scheme 17). Under these CL conditions and in the presence of 1-phenylcycloalkene, no allylic oxidation product was observed. For comparison, the same solution was bubbled with molecular oxygen and irradiated using a Xenon lamp (300 W, λ > 320 nm) [147][148][149], however, no oxidation products were observed. In the absence of TCPO and H 2 O 2 , the corresponding alkene was oxidized leading to the corresponding allylic hydroperoxides via a 1 O 2 oxidation pathway, within 20 minutes (Scheme 17). These results indicate that the observed CL phenomenon didn't induce 1 O 2 production, or the energy transfer and radiative processes of the CL energy acceptors or chemiexcited intermediates are not enough for the oxidation of cycloalkene. Further experiments using different photosensitizers and initial alkenes are in progress to determine this new intermolecular oxidation pathway [150]. the common solvents used in CL procedures, diethylphthalates, the greener ethyl acetate was found to give unexpectedly good results. In addition, several photosensitizers, i.e., Rose Bengal (RB), Methylene Blue, 9,10-diphenylanthracene (9,10-DPA), Eosin Y and Rhodamine B, were tested for light emission through the CL process, with RB providing the best results based on the light emission duration. In the presence of TCPO, H2O2, RB, sodium salicylate and a mixture of ethyl acetate/acetonitrile in a ratio of 2/1, light emission was observed for 40-50 min (Scheme 17). Under these CL conditions and in the presence of 1-phenylcycloalkene, no allylic oxidation product was observed. For comparison, the same solution was bubbled with molecular oxygen and irradiated using a Xenon lamp (300 W, λ > 320 nm) [147][148][149], however, no oxidation products were observed. In the absence of TCPO and H2O2, the corresponding alkene was oxidized leading to the corresponding allylic hydroperoxides via a 1 O2 oxidation pathway, within 20 minutes (Scheme 17). These results indicate that the observed CL phenomenon didn't induce 1 O2 production, or the energy transfer and radiative processes of the CL energy acceptors or chemiexcited intermediates are not enough for the oxidation of cycloalkene. Further experiments using different photosensitizers and initial alkenes are in progress to determine this new intermolecular oxidation pathway [150].
Scheme 17. The CL-induced singlet oxygen production for the oxidation of 1-phenylcyclohexene in "green" solvent ethyl acetate.

Future Prospects
Various technologies have emerged during the past few years aiming to fine-tune the molecular scaffolds and the experimental conditions under which CL can be used for the development of more advanced molecular sensors and new materials, in drug delivery, theranostics and polymerization reactions. To that end, several scaffolds such as diox-Scheme 17. The CL-induced singlet oxygen production for the oxidation of 1-phenylcyclohexene in "green" solvent ethyl acetate.

Future Prospects
Various technologies have emerged during the past few years aiming to fine-tune the molecular scaffolds and the experimental conditions under which CL can be used for the development of more advanced molecular sensors and new materials, in drug delivery, theranostics and polymerization reactions. To that end, several scaffolds such as dioxetanes and luminol-based systems, etc., have already shown great potential and are expected to play a significant role in the years to come. The growing demand for highly efficient PDT platforms that could produce significant amounts of reactive species under CL conditions may also reveal another important research direction that is still unexplored, regarding the replacement of light sources by CL in standard photochemical reactions.
A summary of the major obstacles that cause delay in the expansion of the CL applications include, but are not limited to, (i) the photophysical properties of the CL probe such as the fluorescence quantum yield, emission wavelength, photon flux, CL kinetic profile; (ii) the solubility and stability of the CL probe in the reaction media; (iii) the distance between the energy donor and energy acceptor, plus their emission and absorption spectral overlapping, in the case of direct CL; (iv) the selectivity of the triggering agent and oxidation mechanism of the CL substrates; (v) the efficiency of the emission mechanism (i.e., CIEEL); (vi) the toxicity, when considering bioapplications.
Recently there has been a tremendous increase in the number of new photochemical reactions reported in the literature. Nonetheless, reports of industrial applications utilizing this new knowledge are still scarce and mainly limited to flow-chemistry processes. Maybe nature can inspire the research community to find a solution to this problem through CL?