Recent Advances in Electrochemiluminescence and Chemiluminescence of Metal Nanoclusters

Metal nanoclusters (NCs), including Au, Ag, Cu, Pt, Ni and alloy NCs, have become more and more popular sensor probes with good solubility, biocompatibility, size-dependent luminescence and catalysis. The development of electrochemiluminescent (ECL) and chemiluminescent (CL) analytical methods based on various metal NCs have become research hotspots. To improve ECL and CL performances, many strategies are proposed, from metal core to ligand, from intermolecular electron transfer to intramolecular electron transfer. Combined with a variety of amplification technology, i.e., nanostructure-based enhancement and biological signal amplification, highly sensitive ECL and CL analytical methods are developed. We have summarized the research progresses since 2016. Also, we discuss the current challenges and perspectives on the development of this area.


Introduction
Metal nanoclusters (NCs), comprising only a few to roughly hundreds of metal atoms, have a metal core-protective agent shell structure. Owing to the size of metal NCs approaching the Fermi wavelength of electrons, the spatial confinement of free electrons in metal NCs generates discrete electronic transitions, thereby exhibiting intriguing molecular-like properties. Therefore, metal NCs are deemed to bridge the gap between molecules and nanoparticles. In recent years, they have attracted a great deal of research in the applications of cellular imaging and chemical/biological detection owing to small size, excellent biocompatibility, good stability, distinctive catalytical activity, optical and electrochemical properties [1][2][3][4][5][6][7].
ECL phenomenon of fluorescent Ag NCs was studied for the first time in 2009 [8]. In 2011, Chen's group and Zhu's group found that Au NCs/triethylamine (TEA) system and Au NCs/potassium persulfate system had ECL phenomenon, respectively [9][10][11]. Based on these, they established new methods for the determination of Pb 2+ , dopamine and hydrogen peroxide. Subsequently, Yuan et al. realized the highly sensitive detection of phenols, microRNAs and dopamine with Au, Ag, and Cu NCs as ECL probes and potassium persulfate or hydrazine as coreactants [12][13][14]. Up to now, metal NCs, such as Au, Ag, Cu, Pt, Ni and alloy NCs have been widely studied as luminophores, catalysts, or quenchers in ECL system. Among them, there are many studies on luminophores. Unfortunately, the ECL efficiency of metal NCs is usually low, which significantly limits their sensing capability and application. To solve this problem, many strategies, such as

Au NCs as ECL Luminophores
Au NCs with good water solubility, excellent stability, ease of synthesis and modification have attracted wide interest in ECL field from the fundamental to the analytical applications [47,48]. They have been shown to be prospective nanoscale ECL luminophores that are being extensively explored [49]. However, the relatively low ECL efficiency of Au NCs has been a main obstacle for their better applications. Many efforts from internal metal core to external environment, from intermolecular to intramolecular electron transfer have been made to improve the ECL efficiency.
One important parameter of highly luminescent metal NCs is the valence state of metal atoms in NCs. The reduction degree of Au NCs is positively related to the enhancement of the ECL signals of Au NCs. Peng et al. investigated the valence state effect of Au on the ECL performance of N-acetyl-L-cysteine stabilized Au NCs (NAC/Au NCs) [15]. Once the NAC/Au NCs was reduced by either electrochemical or chemical method, an enhanced ECL was observed. NAC/Au NCs have different valence states of Au, typically as Au(I) and Au(0). Au(I) is responsible for the fluorescence of NAC/Au NCs and Au(0) core is main contribution of NAC/Au NCs in the ECL. Thus, the enhanced ECL signal of electro-reduced NAC/Au NCs was about 30 times higher than that of NAC/Au NCs with K2S2O8 as the coreactant. The ECL quantum efficiency of electro-reduced NAC/Au NCs was 4.11% with [Ru(bpy)3] 2+ as a reference system. This approach had been successfully extended to other Au NCs, such as GSH stabilized Au NCs (GSH/Au NCs). Both the electrochemical and chemical reduction of the GSH/Au NCs showed strong and efficient ECL signals. Therefore, this study presents a new idea to design ECL device from other functional-metal based NCs, but also extends the huge potential application in the ECL sensing. Based on the excellent quenching effect of GSH on the ECL of the NAC/Au NCs, the same group constructed a very simple NAC/Au NCs-based ECL nanosensor for the detection of GSH with a low detection limit of 3.2 × 10 −10 M [50]. More interestingly, the oxidation state of the Au NCs also played a critical role in the ECL. The same group reported a record high ECL quantum yield (ΦECL) of 66% achieved by pre-electrooxidation of L-methionine stabilized Au NCs (Met/Au NCs) with TEA as a coreactant [17]. It was suspected that the electrooxidized product of Met/Au NCs (Met/Au NCs •+ ) could be accumulated on the electrode surface to enhance the ΦECL. This method can be successfully extended to other Au NCs, such as dithiothreitol (DTT) stabilized Au NCs (DTT/Au NCs) and BSA/Au NCs. The ECL signals of pre-oxidized DTT/Au NCs and pre-oxidized BSA/Au NCs enhanced by 20.3-fold

Au NCs as ECL Luminophores
Au NCs with good water solubility, excellent stability, ease of synthesis and modification have attracted wide interest in ECL field from the fundamental to the analytical applications [47,48]. They have been shown to be prospective nanoscale ECL luminophores that are being extensively explored [49]. However, the relatively low ECL efficiency of Au NCs has been a main obstacle for their better applications. Many efforts from internal metal core to external environment, from intermolecular to intramolecular electron transfer have been made to improve the ECL efficiency.
One important parameter of highly luminescent metal NCs is the valence state of metal atoms in NCs. The reduction degree of Au NCs is positively related to the enhancement of the ECL signals of Au NCs. Peng et al. investigated the valence state effect of Au on the ECL performance of N-acetyl-L-cysteine stabilized Au NCs (NAC/Au NCs) [15]. Once the NAC/Au NCs was reduced by either electrochemical or chemical method, an enhanced ECL was observed. NAC/Au NCs have different valence states of Au, typically as Au(I) and Au(0). Au(I) is responsible for the fluorescence of NAC/Au NCs and Au(0) core is main contribution of NAC/Au NCs in the ECL. Thus, the enhanced ECL signal of electro-reduced NAC/Au NCs was about 30 times higher than that of NAC/Au NCs with K 2 S 2 O 8 as the coreactant. The ECL quantum efficiency of electro-reduced NAC/Au NCs was 4.11% with [Ru(bpy) 3 ] 2+ as a reference system. This approach had been successfully extended to other Au NCs, such as GSH stabilized Au NCs (GSH/Au NCs). Both the electrochemical and chemical reduction of the GSH/Au NCs showed strong and efficient ECL signals. Therefore, this study presents a new idea to design ECL device from other functional-metal based NCs, but also extends the huge potential application in the ECL sensing. Based on the excellent quenching effect of GSH on the ECL of the NAC/Au NCs, the same group constructed a very simple NAC/Au NCs-based ECL nanosensor for the detection of GSH with a low detection limit of 3.2 × 10 −10 M [50]. More interestingly, the oxidation state of the Au NCs also played a critical role in the ECL. The same group reported a record high ECL quantum yield (Φ ECL ) of 66% achieved by pre-electrooxidation of L-methionine stabilized Au NCs (Met/Au NCs) with TEA as a coreactant [17]. It was suspected that the electrooxidized product of Met/Au NCs (Met/Au NCs •+ ) could be accumulated on the electrode surface to enhance the Φ ECL . This method can be successfully extended to other Au NCs, such as dithiothreitol (DTT) stabilized Au NCs (DTT/Au NCs) and BSA/Au NCs. The ECL signals of pre-oxidized DTT/Au NCs and pre-oxidized BSA/Au NCs enhanced by 20.3-fold and 5-fold, respectively. BSA is the largest ligand and the electron transfer rate is slowest. Thus, pre-oxidation is more effective for ligand shells, facilitating the electron transfer. This work is an important breakthrough in the ECL field, which not only provides a new method for in-depth study of ECL but also opens up a new avenue for designing and developing high performance ECL probes.
Besides the ECL intensity, the valence states of Au NCs could also affect the ECL wavelength. Kim et al. found that the sources of the ECL and photoluminescent (PL) emissions of the Au NCs were different and the ECL of Au NCs was closely related to the presence of Au(0) in the NCs [51]. Water-soluble GSH/Au NCs reduced by NaBH 4 with TEA as coreactant can generate the unusual near-infrared (NIR) ECL. The research showed that the NIR ECL at 800 nm was ascribed to the Au(0)-GSH motif in the GSH/Au NCs in water, while the PL at 610 nm was attributable to the Au(I)-GSH motif on the surface of GSH/Au NCs. However, Au NCs synthesized by chemical method are usually polydisperse. Most of the studies on the ECL of Au NCs are based on polydisperse Au NCs. In contrast, the synthesis of monodispersed Au NCs of high ECL remains a challenge. The same group further studied the ECL of individual GSH/Au NCs fractionated by gel electrophoresis with TEA as the coreactant [52]. The individual Au NCs possessed unique and significant differences in ECL. When the ratio of Au(I)/Au(0) in the individual Au NCs decreased, NIR ECL of GSH/Au NCs became dominant. This study suggests that the oxidation states of the individual Au NCs will impose significant effect on the ECL wavelength of the clusters. Therefore, the present study can provide a deep insight into the origin of NIR ECL of water-soluble GSH/Au NCs. The findings of Kim and Peng demonstrated that the valence states of Au NCs could both affect the ECL wavelength and intensity of Au NCs.
Furthermore, the change of Au core composition could also affect the ECL signal of Au NCs by the synergistic effect of the two kinds of atoms in NCs core. For example, one study has demonstrated that the doping of Ag into BSA/Au NCs with the molar ratios of 6:1 (Au:Ag) led to the 5-fold ECL emission compared to the individual BSA/Au NCs [38]. A Hg 2+ ECL sensor was developed based on Hg 2+ -induced ECL quenching of BSA/AuAg NCs using TEA as coreactant through the formation of strong metallophilic bonding (Au-Hg 2+ /Ag-Hg 2+ ). The detection limit of Hg 2+ was 5 nM lower than the standard of the World Health Organization for Hg 2+ in drinkable water (30 nM). A similar study also suggested that the ECL signal of GSH stabilized AuAg NCs (GSH/AuAg NCs) was about 100 times higher than that of GSH/Au NCs [40]. The rigid structure of metal core by doping another metal atom could reduce nonradiative transition and thus enhance luminescence of ECL. Chen et al. reported the NIR ECL of rod-shape bimetallic Au 12 Ag 13 NCs [41]. With Ru(bpy) 3 2+ as reference, the self-annihilation ECL of the Au 12 Ag 13 NCs was about 10 times higher. The coreactant ECL of Au 12 Ag 13 NCs was about 400 times stronger than that of Ru(bpy) 3 2+ /tripropylamine (TPrA) system.
The origin of the strong ECL of Au 12 Ag 13 was attributed to the 13th Ag atom at the central position which stabilized the charges on the LUMO orbital and made the rod-shape Au 12 Ag 13 core more rigid. The study could guide the design and syntheses of other NCs or materials in general to achieve improved properties and further affirm the structure-function correlations. The surface ligand of Au NCs plays an important role in ECL reaction. The study found that the ECL intensity of Met/Au NCs was about five times higher than that of BSA/Au NCs with K 2 S 2 O 8 as the coreactant [19]. The oxidized dopamine can lead to the ECL quenching of the Met/Au NCs. The method was used to the detection of dopamine released by cells. The Met/Au NCs showed yellow color luminescence and BSA/Au NCs showed red color luminescence. Therefore, Au NCs-based ECL probes with tunable luminescent properties have potential applications in bioanalysis such as multi-labeling techniques. Yu et al. used the same Met and the similar synthetic method to synthesize Met/Au NCs which demonstrated NIR ECL emission of 835 nm with triethanolamine as coreactant and a 75 times enhanced ECL compared with BSA/Au NCs [53]. As shown in Figure 2, a sandwich-type NIR ECL immunosensor was constructed with the Met/Au NCs as tags and α-fetoprotein (AFP) as a model analyte and exhibited a wide linearity range from 3 fg·mL −1 to 0.1 ng·mL −1 with a detection limit of 1 fg·mL −1 . The ECL enhancement effect of two ligands is better than that of one ligand. Wang's group proposed a novel anodic ECL emitter of thioglycol/glutathione dual ligand-coated Au NCs (TG/GSH/Au NCs) [54]. TG/GSH/Au NCs showed a new ECL peak at a lower potential of + 1.4 V with TEA as coreactant and about 5 times enhanced ECL than that of GSH/Au NCs. Other common thiol compounds had no similar effect.
Inspired by the enhanced PL of Au NCs with a rigid shell, forming rigid host-guest assemblies around Au NCs by introducing another molecule could reduce the non-radiative relaxation of the excited states in ECL processes to enhance the ECL performance of Au NCs. Yang et al. mixed L-arginine (ARG) and 6-aza-2-thiothymine (ATT) protected Au NCs (ATT/Au NCs) to form the rigid host-guest assemblies ARG/ATT/Au NCs via hydrogen bonding [18]. The rigid shell around Au NCs core enhances the radiative-charge-transfer within the Au NCs whilst keeps their excited states unchanged. Thus, the ECL of ARG/ATT/Au NCs was enhanced dramatically than ATT/Au NCs without coreactant and was about 70 times higher to that of ATT/Au NCs with TPrA as coreactant. This study presents a new method for screening novel ECL luminophores with enhanced performance.
The ECL intensity can also be enhanced by using novel coreactants. Wang's group reported the adoption of chelating agent EDTA and commonly used buffer 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) as coreactants for the enhancement of NIR ECL from LA-stabilized Au NCs (LA/Au NCs) at physiological pH [55,56]. Metal ions can modulate ECL signal effectively. Mg 2+ had profound impacts on the ECL features of LA/Au NCs-EDTA system through the complexation with EDTA and the interaction with LA/Au NCs. Zn 2+ was found to enhance the ECL of LA/Au NCs-HEPES system while Mg 2+ and Ca 2+ lowered the ECL signal. The ECL intensity of LA/Au NCs system displayed optimal responses at physiological pH which made HEPES better suited over EDTA for the corresponding applications. The work applies to other ions and other amine-based good's buffers.
The poor conductivity of protein coated on the surface of metal NCs could limit the mass transport and electron transfer, lower the ECL efficiency of NCs and restrict their applications [57]. Coreaction accelerator including some nanoparticle or active micromolecule can improve the ECL emission of NCs through effectively reducing the coreactant to a stronger oxidative or reductive intermediate radical. Jia et al. utilized highly-branched Cu2O as the coreaction accelerator in BSA/AuNCs-K2S2O8 ECL system [58]. Highly-branched Cu2O can catalyze K2S2O8 to produce more radical anion SO4 •− , which can oxidize Au NCs •− to generate more Au NCs*, thus doubling the ECL The ECL enhancement effect of two ligands is better than that of one ligand. Wang's group proposed a novel anodic ECL emitter of thioglycol/glutathione dual ligand-coated Au NCs (TG/GSH/Au NCs) [54]. TG/GSH/Au NCs showed a new ECL peak at a lower potential of + 1.4 V with TEA as coreactant and about 5 times enhanced ECL than that of GSH/Au NCs. Other common thiol compounds had no similar effect.
Inspired by the enhanced PL of Au NCs with a rigid shell, forming rigid host-guest assemblies around Au NCs by introducing another molecule could reduce the non-radiative relaxation of the excited states in ECL processes to enhance the ECL performance of Au NCs. Yang et al. mixed L-arginine (ARG) and 6-aza-2-thiothymine (ATT) protected Au NCs (ATT/Au NCs) to form the rigid host-guest assemblies ARG/ATT/Au NCs via hydrogen bonding [18]. The rigid shell around Au NCs core enhances the radiative-charge-transfer within the Au NCs whilst keeps their excited states unchanged. Thus, the ECL of ARG/ATT/Au NCs was enhanced dramatically than ATT/Au NCs without coreactant and was about 70 times higher to that of ATT/Au NCs with TPrA as coreactant. This study presents a new method for screening novel ECL luminophores with enhanced performance.
The ECL intensity can also be enhanced by using novel coreactants. Wang's group reported the adoption of chelating agent EDTA and commonly used buffer 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) as coreactants for the enhancement of NIR ECL from LA-stabilized Au NCs (LA/Au NCs) at physiological pH [55,56]. Metal ions can modulate ECL signal effectively. Mg 2+ had profound impacts on the ECL features of LA/Au NCs-EDTA system through the complexation with EDTA and the interaction with LA/Au NCs. Zn 2+ was found to enhance the ECL of LA/Au NCs-HEPES system while Mg 2+ and Ca 2+ lowered the ECL signal. The ECL intensity of LA/Au NCs system displayed optimal responses at physiological pH which made HEPES better suited over EDTA for the corresponding applications. The work applies to other ions and other amine-based good's buffers.
The poor conductivity of protein coated on the surface of metal NCs could limit the mass transport and electron transfer, lower the ECL efficiency of NCs and restrict their applications [57]. Coreaction accelerator including some nanoparticle or active micromolecule can improve the ECL emission of NCs through effectively reducing the coreactant to a stronger oxidative or reductive intermediate radical. Jia  produce more cationic radicals TEA •+ which could improve the ECL intensity of BSA/Au NCs-TEA system [59]. The biosensor exhibited ultrahigh immune recognition to procalcitonin with the detection limit as low as 2.36 fg mL −1 . Coreactant accelerator can be also generated in situ. Zhang et al. developed a highly sensitive ECL method for detection of acetylthiocholine (ATCI) based on the catalysis of acetylcholinesterase (AChE) and BSA/AuNCs-K 2 S 2 O 8 ECL system [60]. AChE catalyzed ATCI to produce thiocholine, which acted as the coreactant accelerator to improve the ECL emission. The strategy obtained a low detection limit of 0.17 nM for ATCI. However, the long electron transfer path and great energy loss in the intermolecular ECL reaction between luminophores and coreactants restrict its ECL efficiency [57]. Binary or ternary nanostructure formed by luminophores, coreactants and coreaction accelerators via covalent attachment is benefit for highly efficient ECL [20,46,57,61]. Wang et al. combined LA/Au NCs as the luminophore and N,N-diethylethylenediamine (DEDA) as the coreactant to form the binary nanostructure [20]. The NIR ECL signal of binary nanostructure was about 17 times higher than that from Ru(bpy) 3 2+ /TPrA system.
The design reduced the complication of mass transport between the reactants during the lifetime of radical intermediates involved in conventional ECL generation pathway. The intracluster reactions were highly advantageous for applications by eliminating additional and high excess coreactants otherwise needed. The study opened a new avenue for the designment of high-efficiency Au NCs ECL system. Combining this type of Au NCs ECL system with DNA amplification technology, ECL ultrasensitive detection can be realized. Yang et al. designed a high efficiency ECL label of binary nanostructure with thioctic acid capped Au NCs (TA/Au NCs) as the luminophore and N,N-diisopropylethylenediamine (DPEA) as the coreactant for the ultrasensitive detection of mucin 1 (MUC1) by employing multi-site landing DNA walker as signal amplification strategy [61]. The ECL aptasensor of MUC1 was established with a detection limit of 0.54 fg mL −1 . The study provided a new method for the development of metal NCs-based ultrasensitive ECL platform for biomolecule detection in clinical analysis. In order to further improve ECL efficiency of metal NCs, a highly efficient ECL label containing luminophore, coreactant and coreaction accelerator was designed in a nanostructure [57]. BSA/Au NCs as the luminophore, tris(3-aminoethyl)amine (TAEA) as the coreactant and Pd@CuO nanomaterial as the coreaction accelerator via covalent attachment formed the BSA/Au NCs-TEAE-Pd@CuO ternary nanostructure as highly efficient ECL label for constructing an ultrasensitive biosensor of carcinoembryonic antigen (CEA). The ternary ECL nanostructure showed the maximum ECL intensity and the lowest luminous potential. This indicated that an intramolecular ternary system with faster electronic transfer and more effective energy transmission exhibited optimal ECL performance as compared with others ECL systems. The principle was that both Au NCs and TAEA in nanostructure were oxidized to form Au NCs + -TAEA •+ . Furthermore, the tertiary amine of TAEA lost a proton to generate Au NCs + -TAEA • intermediate. When Pd@CuO as the coreaction accelerator promoted intramolecular electron transfer and energy transmission, more active radical Au NCs + -TAEA • produced the excited state Au NCs*-TAEA, which returned to the ground state and emitted intense light. The detection limit of this method was as low as 16 fg mL −1 which was comparable to other CEA detection literatures. Using the similar design strategy, the same group developed a ECL method for simultaneous detection of CEA and MUC1 based on the BSA/Au NCs with different coreactants and coreaction accelerators for the first time [46]. BSA/Au NCs was chosen as the only luminophore owing to its simultaneous cathodic and anodic ECL emissions under continuous cathodic and anodic scanning. As shown in Figure 3, Au NCs-TiO 2 nanosheets as the cathodic ECL probe exhibited outstanding cathodic ECL signal by combining TiO 2 nanosheets as the coreaction accelerator with O 2 as the coreactant. The ternary nanostructure of BSA/Au NCs-Cu 2 O@Cu nanoparticles-N,N-diethylethylenediamine (BSA/Au NCs-Cu 2 O@Cu NPs-DEDA) acted as the anodic ECL probe to obtain excellent anodic ECL emission. On the basis of cathodic and anodic Au NCs nanocomposites as high-efficient ECL probes and target-catalyzed hairpin hybridization as signal amplification strategy, an ultrasensitive ECL aptasensor was constructed for simultaneous detection of CEA and MUC1, with detection limits of 5.8 fg mL −1 and 0.43 pg mL −1 , respectively. The proposed strategy easily solved one main technical challenge of cross reactions of dual-luminophores for dual-biomarker detection and realized single-step detection of dual biomarkers simultaneously, which initiated a new thought in realizing a new generation of dual-biomarker ECL detection beyond the traditional ones in sensing analysis and diagnostic sensing.
Molecules 2020, 25, x FOR PEER REVIEW 7 of 23 0.43 pg mL −1 , respectively. The proposed strategy easily solved one main technical challenge of cross reactions of dual-luminophores for dual-biomarker detection and realized single-step detection of dual biomarkers simultaneously, which initiated a new thought in realizing a new generation of dual-biomarker ECL detection beyond the traditional ones in sensing analysis and diagnostic sensing. Guided by the design principles toward AIE, the AIE characteristics of the metal NCs were found [43,44]. Drying 6-aza-2-thiothymine (ATT) protected Au NCs (ATT/Au NCs) on glass carbon electrodes enhanced emission by aggregation-induced ECL (AIECL) with TEA as the coreactant [43]. The quantum yield of ATT/Au NCs in TEA was 0.8%, while dried ATT/Au NCs was 78% with Ru(bpy)3 2+ as a reference. It was speculated that drying process effectively suppresses the vibration and rotation of ATT and thus the nonradiative relaxation. Thus, the ECL quantum yield of ATT/Au NCs was enhanced significantly. Besides drying, aggregation of Au NCs caused by metal ions can also improve ECL efficiency. Wang et al. investigated the anodic ECL features of adenosine triphosphate (ATP) protected Au NCs (ATP/Au NCs) before and after Ca 2+ induced aggregation [44]. The ECL of ATP/Au NCs increases significantly 50-fold after adding Ca 2+ . TEM clearly showed the aggregated rod-like ATP/Au NCs assembly caused by the strong binding of Ca 2+ to phosphate group on the ATP. It was indicated that the AIECL behaviors occurred. A calmodulin ECL sensor was developed based on the binding of Ca 2+ to calmodulin with a limit of detection 0.1 μg mL −1 . Other strategies of nanostructure substrate amplification effect, biological amplification effect, ECL resonance energy transfer and synergistic effect are also used to improve the ECL performances of Au NCs [62][63][64]. Guided by the design principles toward AIE, the AIE characteristics of the metal NCs were found [43,44]. Drying 6-aza-2-thiothymine (ATT) protected Au NCs (ATT/Au NCs) on glass carbon electrodes enhanced emission by aggregation-induced ECL (AIECL) with TEA as the coreactant [43]. The quantum yield of ATT/Au NCs in TEA was 0.8%, while dried ATT/Au NCs was 78% with Ru(bpy) 3 2+ as a reference. It was speculated that drying process effectively suppresses the vibration Though Au NCs are observed to show high ECL intensity commonly used in ECL systems, the ECL luminescent properties of other metal NCs have been gradually explored, such as Ag, Cu, Pt and Ni NCs. At present, the methods to improve the ECL luminescence intensity of Ag NCs are to design DNA structure, improve the stability and uniformity of silver nanoclusters synthesis, and enhance the ECL signal; or to combine nucleic acid amplification technology, co-reaction accelerator or surface plasmon resonance (SPR) of metal nanoparticles to increase ECL.
DNA is the most commonly used template for Ag NCs synthesis. The ECL properties of Ag NCs are highly dependent on the nature of the template molecules. Resolving their stability and homogenous questions are quite attractive for the enhancement of ECL efficiency of Ag NCs. Feng's team used a triplex DNA template for the synthesis of site-specific, homogeneous and highly stable Ag NCs [65]. Ag NCs were formed in the very position of CG.C + triplet site in triplex DNA. As the number of CG.C + was more, the ECL intensity of Ag NCs was stronger. A strong ECL signal was observed for the triplex DNA-Ag NCs solution, while comparable weak ECL was observed when duplex DNA and single strand DNA used as templates. A signal-on pattern of ECL sensor was developed for the detection of biothiols based on the enhanced catalytic reaction and a robust interaction between the triplex-AgNCs and cysteine, by influencing the microenvironment provided by DNA template. The triplex-templated Ag NCs would be promising candidates in the ECL analysis with the good stability and easier preparation.
The combination of ECL of Ag NCs with the nucleic acid based biological amplification strategy is another common ECL enhancement method. Jie's group developed a highly sensitive Ag NCs for thrombin detection based on DNAzyme-assisted target recycling and hybridization chain reaction (HCR) multiple amplification strategy [66]. As shown in Figure 4, thrombin recognition opened the hairpin DNA and initiated the binding of the substrate with the DNAzyme (red line). The fragment of single-stranded DNA (ssDNA) was released followed by hydrolytic cleavage with catalytic effect of Zn 2+ . This induced the DNAzyme recycling and generation of a large amount fragment of ssDNA as target DNA to execute more reactions. The introduced H1 and H2 can trigger the HCR process to form linear dsDNA with cytosine-rich templates, which facilitated the in-situ syntheses of Ag NCs as ECL and electrochemical tags. The detection limit of thrombin was as low as 0.1 fM, showing potential of the method for other biomolecules detection.
In addition to nucleic acid signal amplification, it can also be combined with coreaction accelerators to enhance ECL. Using in situ formed Ag NCs and A novel mechanism named surface plasmon-enhanced ECL was found to drastically enhance the ECL of Ag NCs. Wang et al. fabricated a high sensitivity and selectivity miRNA-21 biosensor based on surface plasmon-enhanced ECL with DNA templated Ag NCs as ECL luminophores and Au nanoparticles (Au NPs) as the localized surface plasmon resonance source [68]. The ECL enhancement of Ag NCs by Au NPs is related with the separation distance between Ag NCs and Au NPs and the electrodeposition time of Au NPs. Combined a cyclic amplification process with enzyme-free catalytic hairpin DNA, miRNA-21 was successfully detected with a wide linear range from 1 aM to 10 4 fM and a relatively low detection limit of 0.96 aM.
Considering the high consumption of noble gold and silver, Cu NCs as a nontoxic and low-cost candidate, have gained growing interest in ECL applications. The anodic and blue ECL of the Cu NCs with hydrazine as coreactant for the determination of dopamine was first demonstrated by Yuan's group [14]. However, the electron-transfer path between the Cu NCs in aqueous and the sensing interface was so long that much energy was lost and the ECL luminous of Cu NCs was inhibited. In addition, the tight molecular arrangement of Cu NCs stabilized by BSA would increase the inner-filter effect and impede the electrochemical activation of the internal ECL emitters. Therefore, the sequence-dependent synthesis of Cu NCs via the stable A-Cu 2+ -T bond was developed [69]. The longitudinal distribution of Cu NCs could be controlled onto DNA templates and the lateral distance could be designed by the DNA nanocrane. Based on this, the same group developed two kinds of highly sensitive Cu NCs for miRNA detection. One was based on the exonuclease (III-assisted amplification, HCR process and TiO 2 as the coreaction accelerator [70]. The other one was based on the DNA nanocrane with binding-induced DNA assembly as manipulator and tetrahedral DNA nanostructure as base ( Figure 5) [71]. This study had three novel ideas. First, the binding-induced DNA assembly finely manipulated the generation of AT-rich dsDNA. On the basis of the stable A-Cu 2+ -T, the DNA nanocranes realized a small quantity of miRNA-155 that triggered the generation of a large amount of Cu NCs. Second, TDN could not only improve the probe spacing to generate more Cu NCs but also lessen collision annihilation of excited-state species Cu NCs* for retaining remarkable ECL emission, which significantly promoted the sensitivity of the biosensor. Third, the reasonably designed biosensor simultaneously realized activity modulation of ECL probes (the DNA-stabilized Cu NCs) and target recycling amplification, achieving ultrasensitive analysis of miRNA-155 with the detection limit of 36 aM. With the ingenious combination of DNA nanocranes, the proposed biosensor exhibited excellent selectivity, accuracy, and stability and provided a dynamic sensing platform for various target analysis in diverse applications, such as early disease diagnosis and environmental analysis.
NCs are highly dependent on the nature of the template molecules. Resolving their stability and homogenous questions are quite attractive for the enhancement of ECL efficiency of Ag NCs. Feng's team used a triplex DNA template for the synthesis of site-specific, homogeneous and highly stable Ag NCs [65]. Ag NCs were formed in the very position of CG.C + triplet site in triplex DNA. As the number of CG.C + was more, the ECL intensity of Ag NCs was stronger. A strong ECL signal was observed for the triplex DNA-Ag NCs solution, while comparable weak ECL was observed when duplex DNA and single strand DNA used as templates. A signal-on pattern of ECL sensor was developed for the detection of biothiols based on the enhanced catalytic reaction and a robust interaction between the triplex-AgNCs and cysteine, by influencing the microenvironment provided by DNA template. The triplex-templated Ag NCs would be promising candidates in the ECL analysis with the good stability and easier preparation.
The combination of ECL of Ag NCs with the nucleic acid based biological amplification strategy is another common ECL enhancement method. Jie's group developed a highly sensitive Ag NCs for thrombin detection based on DNAzyme-assisted target recycling and hybridization chain reaction (HCR) multiple amplification strategy [66]. As shown in Figure 4, thrombin recognition opened the hairpin DNA and initiated the binding of the substrate with the DNAzyme (red line). The fragment of single-stranded DNA (ssDNA) was released followed by hydrolytic cleavage with catalytic effect of Zn 2+ . This induced the DNAzyme recycling and generation of a large amount fragment of ssDNA as target DNA to execute more reactions. The introduced H1 and H2 can trigger the HCR process to form linear dsDNA with cytosine-rich templates, which facilitated the in-situ syntheses of Ag NCs as ECL and electrochemical tags. The detection limit of thrombin was as low as 0.1 fM, showing potential of the method for other biomolecules detection. Recombination(composition) and doping are also the methods to improve the ECL efficiency of Cu NCs [72,73]. Liu et al. combined dithiothreitol functioned Cu NCs (DTT/CuNCs) and electrocatalyst material carbon nitride nanosheets (CNNSs) to form a nanocomposite material (DTT/Cu NCs/CNNSs) [72]. The pure CNNSs delivered high but unstable ECL intensity performance and the ECL intensity of DTT/Cu NCs was the weakest with K 2 S 2 O 8 as coreactant. But the DTT/Cu NCs/CNNSs nanocomposite provided the excellent and reasonably stable ECL signal. Hg 2+ can chelate the free SH groups on the surface of DTT/Cu NCs and suppress the formation of anion-radicals of DTT/Cu NCs/CNNSs. The method was successfully applied to the detection of Hg 2+ in water pollution monitoring with a detection limit of 0.01 nM. Doping the rich electronic rare earth element into Cu NCs is another effectively method to improve the stability of Cu NCs and to increase ECL intensity.
Zhuang et al. studied the ECL enhancement phenomenon of Eu 3+ ion doped GSH stabilized Cu NCs (GSH/Cu NCs) for the first time [73]. Eu 3+ ions can alter the surfaces of Cu NCs and drive the formation of new surface states (Eu(III) complex) to promote effective energy transfer from the host to the Eu 3+ ions. The ECL intensity of Eu 3+ -GSH/Cu NCs increased by 3-fold and the stability of GSH/Cu NCs was enhanced greatly. The method provided a simple mode for turn-off detection of dopamine with a detection limit of 1.0 × 10 −11 M. The presented results give us a clue to overcome the stability issues of metal NCs, provide a powerful tool for the development of ECL emitters, and may open up promising avenues to develop new ECL systems for medical and biological analysis.  Unusual Pt NCs and Ni NCs also exhibit ECL properties [74,75]. Graphene (GR) could immobilize Pt NCs and accelerate electron transfer between the electrode and coreactant TEA which enhanced the ECL intensity of GR/Pt NCs two times than that of Pt NCs [74]. Babamiri et al. developed a novel molecularly imprinted polymer (MIP)-ECL sensor for selective detection of creatinine based on BSA/Ni NCs as emitter and tri-n-propylamine as coreactant [75]. The ITO electrode was modified with a magnetic graphene oxide MIP film with BSA/Ni NCs-embedded in MIP. In the presence of creatinine, the ECL emission of BSA/Ni NCs was strongly quenched owing to the imprinted cavities of MIP film occupied.

Metal NCs as ECL Quenchers
Generally, the luminous efficiency of quantum dots (QDs) is higher than that of metal NCs. When the absorption peak of the metal NCs matches the ECL emission peak of the QDs, ECL resonance energy transfer between the QDs and metal NCs can be performed. There are few reports on this aspect, some of which have been summarized in the previous review [45]. Recently, Jie et al. designed a versatile ECL method for detection of thrombin and microRNA-21 based on the Ag(I) ion-enhanced or Ag NCs quenched ECL of CdSe QDs [76]. Bipedal molecular machine (BMM)-triggered surface programmatic chain reaction (SPCR) coupled with mesoporous silica nanoparticle (MSN) multiple amplification is used to introduce plentiful QDs and Ag + ions to Unusual Pt NCs and Ni NCs also exhibit ECL properties [74,75]. Graphene (GR) could immobilize Pt NCs and accelerate electron transfer between the electrode and coreactant TEA which enhanced the ECL intensity of GR/Pt NCs two times than that of Pt NCs [74]. Babamiri et al. developed a novel molecularly imprinted polymer (MIP)-ECL sensor for selective detection of creatinine based on BSA/Ni NCs as emitter and tri-n-propylamine as coreactant [75]. The ITO electrode was modified with a magnetic graphene oxide MIP film with BSA/Ni NCs-embedded in MIP. In the presence of creatinine, the ECL emission of BSA/Ni NCs was strongly quenched owing to the imprinted cavities of MIP film occupied.

Metal NCs as ECL Quenchers
Generally, the luminous efficiency of quantum dots (QDs) is higher than that of metal NCs. When the absorption peak of the metal NCs matches the ECL emission peak of the QDs, ECL resonance energy transfer between the QDs and metal NCs can be performed. There are few reports on this aspect, some of which have been summarized in the previous review [45]. Recently, Jie et al. designed a versatile ECL method for detection of thrombin and microRNA-21 based on the Ag(I) ion-enhanced or Ag NCs quenched ECL of CdSe QDs [76]. Bipedal molecular machine (BMM)-triggered surface programmatic chain reaction (SPCR) coupled with mesoporous silica nanoparticle (MSN) multiple amplification is used to introduce plentiful QDs and Ag + ions to significantly improve the ECL signal for sensitive "signal on" detection of thrombin. Due to the ECL resonance energy transfer (RET) between Ag NCs acceptors and CdSe QD donors, the ECL signal of CdSe QDs was quenched. In addition, Ag NCs competitively reacted with S 2 O 8 2− to reduce the production of ECL co-reactive free radicals (SO 4 −• ), which further decreased the ECL signal; thus a "signal off" ECL biosensor for ultrasensitive detection of microRNA-21 was developed ( Figure 6). Yang et al. designed a wavelength-resolved ECL resonance energy transfer (ECL-RET) ratiometric immunosensor from Au NPs functionalized graphite-like carbon nitride nanosheets (Au-g-C 3 N 4 ) to Au NCs [77]. The well-matched ECL emission spectrum of Au-g-C 3 N 4 and absorption spectrum of Au NCs, as well as the absence of ECL emission of Au NCs, make the system an efficient and interference-free ECL-RET ratiometric system. As a consequence, a sensitive ECL biosensor for detection of cardiac troponin I was accomplished with a wide linear range from 50 fg mL −1 to 50 ng mL −1 and a low detection limit of 9.73 fg mL −1 . This work enriched the wavelength-resolved ECL-RET system and provided an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.
Molecules 2020, 25, x FOR PEER REVIEW 11 of 23 significantly improve the ECL signal for sensitive ''signal on'' detection of thrombin. Due to the ECL resonance energy transfer (RET) between Ag NCs acceptors and CdSe QD donors, the ECL signal of CdSe QDs was quenched. In addition, Ag NCs competitively reacted with S2O8 2− to reduce the production of ECL co-reactive free radicals (SO4 − • ), which further decreased the ECL signal; thus a "signal off" ECL biosensor for ultrasensitive detection of microRNA-21 was developed ( Figure 6). Yang et al. designed a wavelength-resolved ECL resonance energy transfer (ECL-RET) ratiometric immunosensor from Au NPs functionalized graphite-like carbon nitride nanosheets (Au-g-C3N4) to Au NCs [77]. The well-matched ECL emission spectrum of Au-g-C3N4 and absorption spectrum of Au NCs, as well as the absence of ECL emission of Au NCs, make the system an efficient and interference-free ECL-RET ratiometric system. As a consequence, a sensitive ECL biosensor for detection of cardiac troponin I was accomplished with a wide linear range from 50 fg mL −1 to 50 ng mL −1 and a low detection limit of 9.73 fg mL −1 . This work enriched the wavelength-resolved ECL-RET system and provided an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.

Metal NCs as ECL Catalysts
Luo et al. proposed a sensitive ECL biosensor for detection of protein kinase activity (PKA) based on the BSA/Au NCs enhanced cathodic ECL of graphite-like carbon nitride material (g-C3N4)-S2O8 2− system [78]. After dropping the g-C3N4 onto the surface of a glassy carbon electrode (GCE), peptides were assembled by reaction with chitosan. Using PKA as a model kinase, BSA/Au NCs were immobilized onto the phosphorylated peptides modified g-C3N4/GCE by Au-S bond in the presence of adenosine 5′-[γ-thio] triphosphate (ATP-s) and PKA. The assembled BSA/Au NCs as the catalysts of the cathode ECL reaction of g-C3N4 can significantly enhance the ECL intensity. The QDs as donors. Reprinted from [76] with permission from RSC.

Metal NCs as ECL Catalysts
Luo et al. proposed a sensitive ECL biosensor for detection of protein kinase activity (PKA) based on the BSA/Au NCs enhanced cathodic ECL of graphite-like carbon nitride material (g-C 3 N 4 )-S 2 O 8 2− system [78]. After dropping the g-C 3 N 4 onto the surface of a glassy carbon electrode (GCE), peptides were assembled by reaction with chitosan. Using PKA as a model kinase, BSA/Au NCs were immobilized onto the phosphorylated peptides modified g-C 3 N 4 /GCE by Au-S bond in the presence of adenosine 5 -[γ-thio] triphosphate (ATP-s) and PKA. The assembled BSA/Au NCs as the catalysts of the cathode ECL reaction of g-C 3 N 4 can significantly enhance the ECL intensity. The resultant ECL signal of g-C 3 N 4 was magnified 4.5 times compared with the absence of BSA/Au NCs. The proposed ECL platform is capable of analysing PKA activity of biological sample quantitatively and screening kinase inhibition qualitatively.
Pt NCs play double roles of the acceptor and the donor in the multiple ECL resonance energy transfer (RET) system [79]. The PL emission spectrum of the Alexafluor (AF) overlapped with both the UV-Vis absorption spectra of polyethyleneimine stabilized Pt NCs and tris(4,4 -dicarboxylic acid-2,2 -bipyridyl) ruthenium(II) dichloride (Ru(dcbpy) 3 2+ ). It is indicated that resonance energy transfer (RET) can be generated not only between AF and Pt NCs but also between AF and Ru(dcbpy) 3 2+ .
Then the PL emission spectrum of the Pt NCs overlaps with the UV-vis absorption spectrum of Ru(dcbpy) 3 2+ , displaying the presence of RET between Pt NCs and Ru(dcbpy) 3 2+ . Therefore Pt NCs can accept energy from alexafluor, transfer it to Ru(dcbpy) 3 2+ and then enhance ECL efficiency.
The existence of multiple energy donor/acceptor pairs in the same nanostructure resulted in a shorter electron-transfer path, less energy loss and higher RET efficiency. Combined with target recycling amplification technology, the ECL efficiency is 1.78 times higher than the classic Ru(bpy) 3 2+ . From what has been discussed above, a brief summary of the methods to improve ECL efficiency of metal NCs was shown in Table 1. Ligand New Ligand L-methionine stabilized Au NCs + K 2 S 2 O 8 [19] L-methionine stabilized Au NCs + triethanolamine [53] Double Ligand Thioglycol/glutathione dual ligand-coated Au NCs + TEA [54] Rigid Host-Guest Assemblies Mixed L-arginine (ARG) and 6-aza-2-thiothymine (ATT) protected Au NCs + TPrA [18] External environment Novel coreactants LA-stabilized Au NCs / EDTA [55] LA-stabilized Au NCs / HEPES [56] Coreaction accelerator BSA/AuNCs + K 2 S 2 O 8 , highly-branched Cu 2 O as the coreaction accelerator [58] BSA/AuNCs + TEA, Cu 2 S snowflake as the coreaction accelerator [59] BSA/AuNCs + K 2 S 2 O 8 , thiocholine produced in situ as the coreaction accelerator [60] Intramolecular Electron Transfer: Binary or ternary nanostructure formed by luminophores, coreactants and coreaction accelerators via covalent attachment Binary nanostructure formed by LA/Au NCs as the luminophore and DEDA as the coreactant [20] Binary nanostructure formed by TA/Au NCs as the luminophore and DPEA as the coreactant [61] Ternary nanostructure formed by BSA/Au NCs as the luminophore, TAEA as the coreactant and Pd@CuO nanomaterial as the coreaction accelerator [57] Binary nanostructure formed by BSA/Au NCs as the luminophore, TiO 2 nanosheets as the coreaction accelerator with O 2 as the coreactant.
[46] Ternary nanostructure of BSA/Au NCs-as the luminophore, Cu 2 O@Cu nanoparticles as the coreaction accelerator, and DEDA as the coreactant.

The Catalysis of Single Metal NCs
Chen et al. found that BSA/Au NCs can enhance the intensity of CL system, such as K 3 Fe(CN) 6  Other template stabilized Au NCs also have catalytic effect on some CL systems. Hemoglobin-stabilized Au NCs displayed a strong catalytic effect on the luminol-NaIO 4 system [91].
Dopamine can be used as scavenging agents of reactive oxygen species and then inhibited the CL signal. This method was used for the measurement of dopamine in human plasma. Li et al. developed a CL method for detection of kanamycin based on DNA templated Au NCs catalysis and the separation of magnetic beads [84]. As shown in Figure 7, the strategy designed two single-stranded DNAs (DNA1 and DNA2). DNA1 comprised the aptamer of kanamycin. DNA2 consisted of repeat adenosine bases to prepare Au NCs by UV-light-assisted method and a nucleotide sequence to hybridize with DNA1. This strategy provided a versatile method for detection of different targets by altering the aptamer sequence of DNA1.  The catalytic activity of BSA stabilized Ag NCs (BSA/Ag NCs) and BSA stabilized Cu NCs (BSA/Cu NCs) in luminol-H2O2 system has also been reported [82,85]. Fan et al. firstly prepared four kinds of BSA/Ag NCs using different amount of BSA. BSA/Ag NCs with less BSA had higher affinity to H2O2 and were beneficial to the generation of • OH and 1 O2 [82]. The determination of H2O2 was allowed. H2O2 is the oxidative product of uric acid in the presence of the urate oxidase. CL detection of uric acid was realized with a detection limit of 0.75 μM. Using the similar design strategy, Xu et al. developed a sensitive CL method for detection of cholesterol in milk and human serum samples based on the catalytic activity of BSA/Cu NCs [85]. These works can be applied to the detection of other enzyme-coupled catalyzed biologically molecules.
Cysteine capped Cu NCs (Cys/Cu NCs) also strongly enhanced the CL signal of luminol-H2O2 reaction [86] and diperiodatoargentate (III)-folic acid reaction [88]. Phenylalanine and tryptophan could enhance the sensitivity of Cys/Cu NCs-luminol-H2O2 system. Nitrite inhibited CL intensity of Cys/Cu NCs-diperiodatoargentate (III)-folic acid system. Benefiting from these phenomena, the sensors were available for detection of phenylalanine and tryptophan in human serum, and folic acid and nitrite in water, pickled vegetable and sausage samples with satisfactory recoveries.

Methods for Improving Catalytic Efficiency of Metal NCs
In order to further improve the catalytic efficiency of metal NCs, four main strategies are proposed so far, such as cationic metal NCs, metal NCs-MOF structure (metal-organic frameworks, MOFs), bimetal core NCs and synergistic effect of nanomaterials. Cationic Au NCs was designed based on ethanediamine modified on the surface of BSA/Au NCs [83]. The cationic BSA/Au NCs showed higher catalytic activity in the luminol-H2O2 system than did unmodified Au NCs. The principle was based on the generation of 1 O2 on BSA/Au NCs cationic surface and better affinity between luminol and cationic BSA/Au NCs. The cationic BSA/Au NCs were believed as an artificial The catalytic activity of BSA stabilized Ag NCs (BSA/Ag NCs) and BSA stabilized Cu NCs (BSA/Cu NCs) in luminol-H 2 O 2 system has also been reported [82,85]. Fan et al. firstly prepared four kinds of BSA/Ag NCs using different amount of BSA. BSA/Ag NCs with less BSA had higher affinity to H 2 O 2 and were beneficial to the generation of • OH and 1 O 2 [82]. The determination of H 2 O 2 was allowed. H 2 O 2 is the oxidative product of uric acid in the presence of the urate oxidase. CL detection of uric acid was realized with a detection limit of 0.75 µM. Using the similar design strategy, Xu et al. developed a sensitive CL method for detection of cholesterol in milk and human serum samples based on the catalytic activity of BSA/Cu NCs [85]. These works can be applied to the detection of other enzyme-coupled catalyzed biologically molecules.
Cysteine capped Cu NCs (Cys/Cu NCs) also strongly enhanced the CL signal of luminol-H 2 O 2 reaction [86] and diperiodatoargentate (III)-folic acid reaction [88]. Phenylalanine and tryptophan could enhance the sensitivity of Cys/Cu NCs-luminol-H 2 O 2 system. Nitrite inhibited CL intensity of Cys/Cu NCs-diperiodatoargentate (III)-folic acid system. Benefiting from these phenomena, the sensors were available for detection of phenylalanine and tryptophan in human serum, and folic acid and nitrite in water, pickled vegetable and sausage samples with satisfactory recoveries.

Methods for Improving Catalytic Efficiency of Metal NCs
In order to further improve the catalytic efficiency of metal NCs, four main strategies are proposed so far, such as cationic metal NCs, metal NCs-MOF structure (metal-organic frameworks, MOFs), bimetal core NCs and synergistic effect of nanomaterials. Cationic Au NCs was designed based on ethanediamine modified on the surface of BSA/Au NCs [83]. The cationic BSA/Au NCs showed higher catalytic activity in the luminol-H 2 O 2 system than did unmodified Au NCs. The principle was based on the generation of 1 O 2 on BSA/Au NCs cationic surface and better affinity between luminol and cationic BSA/Au NCs. The cationic BSA/Au NCs were believed as an artificial peroxidase for potential applications.
In order to improve the stability and catalytic efficiency of Cu NCs, Nano-pores of CuMOFs had been exploited as hosts for encapsulating Cu NCs to form Cu NCs@CuMOFs nanocomposite [89]. The nanocomposite enhanced the CL emission of KMnO 4 -rhodamine 6 G reaction by about 48 times, which was greater than bare Cu NCs (just 15 times). Tramadol could enhance the CL emission of KMnO 4 -rhodamine 6 G-Cu NCs@CuMOFs system. This method displayed good analytical performance with linear range from 0.0030 µM to 2.5 µM, with a detection limit of 0.80 nM. They also proposed that synergetic catalytic effects of BSA/Au NCs-graphene quantum dots (GQDs) and BSA/Ag NCs-GQDs enhanced the weak emission of KMnO 4 -rhodamine B CL reaction [31,32]. Cimetidine can decrease the CL intensity of KMnO 4 -rhodamine B-BSA/Au NCs-GQDs when interacting with these catalysts and reducing their activity. Rabeprazole showed a selective enhancing impact on the KMnO 4 -rhodamine B-BSA/Ag NCs-GQDs CL emission. The method was used for the detection of rabeprazole in human urine. Compared

Metal NCs as CL Luminophores
Compared with reports of the catalytic action of metal NCs, the investigation of metal NCs as luminophores of CL is rare. Li et al. designed a CL resonance energy transfer platform for sensitive and label-free detection of trypsin [35]. Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H 2 O 2 acted as energy donor and BSA/Au NCs acted as energy acceptor. The BSA/Au NCs produced intense CL by accepting the energy from TCPO-H 2 O 2 CL reaction. The CL intensity of BSA/Au NCs decreased when BSA was cleft by trypsin. As a consequence, a sensitive CL method for detection of trypsin was accomplished with a wide linear range from 0.01 µg mL −1 to 50.0 µg mL −1 with a detection limit of 9 ng mL −1 . They continued to research direct CL of BSA/Au NCs with classic oxidants, such as KMnO 4 , N-bromosuccinimide, K 3 Fe(CN) 6 , H 2 O 2 and Ce(IV) [34]. The highest CL signal was from acidic KMnO 4 -BSA/Au NCs CL reaction. The possible luminophore was the excited state Mn(II) * , originating from the reduction of KMnO 4 with BSA/Au NCs. H 2 O 2 can decreased the CL signal of acidic KMnO 4 -BSA/Au NCs system. Hence, the novel CL system was developed for the H 2 O 2 determination with a linear range from 1.0 × 10 −6 mol L −1 to 1.0 × 10 −4 mol L −1 . Cys/Cu NCs also played the role of reductant in the KMnO 4 -Cys/Cu NCs CL system [33]. Luminophore was the excited state Mn(II) * , originating from the reduction of KMnO 4 with Cys/Cu NCs. Yuan et al. utilized hyperbranched polyethyleneimine (hPEI) as a template for the synthesis of hPEI/Ag NCs, which emitted CL by reacting with hydroxy radicals [36]. The emission originated from excited hPEI/Ag NCs. The molecular weight of hPEI affected the CL signal of hPEI/Ag NCs and 10 K hPEI/Ag NCs displayed the strongest CL signal. Tea polyphenols served as antioxidants consumed hydroxy radicals and resulted in a decrease in the CL signal. A sensitive detection method for tea polyphenols was developed based on this phenomenon. The excited state of Cys/Cu NCs also acted as a luminophore in the Ce(IV)-Cu NCs CL system [33].

Metal NCs as CL Quenchers
There are few reports on quenching CL of metal NCs in recent five years. Vahid et al. found that CdSe quantum dots (CdSe QDs) increased the CL intensity of H 2 O 2 -HCO 3 − system based on the CL resonance energy transfer (CRET) between the CL emitters and CdSe QDs and the catalytic activity of CdSe QDs [37]. BSA/Au NCs could prohibit the CRET system and turn off the CL emission. The CL signal was recovered because of the leaching effect of cyanide on Au NCs. This strategy resulted in a highly sensitive and reliable measurement of cyanide in environmental waters and biological samples.

Summary and Future Perspectives
In the past five years, tremendous literatures on the applications of metal NCs have been reported. In this presented review, we introduce recent research progress in the ECL and CL of metal NCs. Metal NCs with novel catalytic, electrical and optical properties can be obtained on various thiols, protein, DNA, polymer through chemical and electrochemical methods simply, rapidly and cheaply. On account of the advantages of metal NCs, such as small size, good biocompatibility, and luminosity, metal NCs gradually become a multipurpose tool promising for applications in sensing of DNAs and RNAs, metal ions, proteins and enzymes, small biomolecules and so on. This review focuses on the roles of metal NCs in CL and ECL fields, such as luminophores, catalysts, and quenchers. Many efforts have been devoted to enhance the ECL efficiency of metal NCs and the catalytic efficiency of metal NCs in CL system. Some recently reported detection methods using metal NCs were listed in the Table 2. Met/Au NCs + triethanolamine α-Fetoprotein 3 fg mL −1 -0.1 ng mL −1 1 fg mL −1 [53] BSA/Au NCs + TAEA CEA 100 fg mL −1 -100 ng mL −1 16 fg mL −1 [57]   However, a great deal of challenges and difficulties remain in this exciting field of science. First, the chemical synthesis of metal NCs produces atomically precise but polydisperse clusters. In addition, the stability and dispersion of metal NCs has a certain influence on the luminous efficiency and catalytic efficiency. Therefore, it is urgent to explore new synthesis and purification methods to get metal NCs with single size, good stability and dispersion. Both Cu NCs and Ag NCs can improve their dispersion and control the size by designing DNA structures. DNA synthesis of Au NCs and other metal NCs has not been reported. Protein is one of the main protectants for the synthesis of metal NCs. Can peptides replace proteins in the synthesis of metal NCs? Can the size, dispersion and stability of metal NCs be improved if nanomaterials with special structures are introduced, such as MOF materials? These are all questions worth studying.
Second, although many kinds of metal NCs have been studied, such as Au, Ag, Pt, Cu, Ni, and some alloy NCs. It is still one of the directions to develop other non-noble metal NCs, alloy NCs or doped NCs to reduce costs and develop new properties. At present, the metal cores of alloy nanoclusters are mainly composed of two kinds of metals. There have been no clusters of three or more metallic elements. Doped elements are also very limited, electron-rich element doping may be one direction.
Third, the principles of metal NCs as luminescent agents, catalysts or quenchers are very complex. It is very important to study systematically the influence of metal nucleus, ligand, structure, solvent and charge on the properties of metal NCs. For example, whether the valence state-dependent ECL properties of the Au NCs can also be applied to other metal NCs? What about intramolecular electron transfer and aggregation-induced ECL? Only when one principle or strategy is verified on a variety of metal nanoclusters, its rationality can be effectively explained.
It is expected that with the recognition of the advantages of metal NCs by more researchers, emerging excellent work will be reported in the near future.