Recent advances in the analytical applications of copper nanoclusters

Accepted Manuscript Title: Recent advances in the analytical applications of copper nanoclusters Author: Xue Hu, Tingting Liu, Yunxia Zhuang, Wei Wang, Yinying Li, Wenhong Fan, Yuming Huang PII: DOI: Reference:

S0165-9936(15)30180-1 http://dx.doi.org/doi: 10.1016/j.trac.2015.12.013 TRAC 14622

To appear in:

Trends in Analytical Chemistry

Please cite this article as: Xue Hu, Tingting Liu, Yunxia Zhuang, Wei Wang, Yinying Li, Wenhong Fan, Yuming Huang, Recent advances in the analytical applications of copper nanoclusters, Trends in Analytical Chemistry (2016), http://dx.doi.org/doi: 10.1016/j.trac.2015.12.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Recent advances in the analytical applications of copper nanoclusters Xue Hua, Tingting Liua, Yunxia Zhuanga, Wei Wanga, Yinying Lia, Wenhong Fanb,, Yuming Huanga, a

Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education,

College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China b

School of Chemistry and Environment, Beihang University, Beijing 100191, China

HIGHLIGHTS 

Copper nanoclusters (Cu NCs) possess unique chemical properties for sensing



Progress in the analytical applications of Cu NCs is amazing



Analytical applications of Cu NCs are reviewed

ABSTRACT Metal nanoclusters (MNCs) are composed of several to tens of atoms and have drawn considerable research interest due to their unique electrical, physical and optical properties. However, in comparison to the extensively investigated Au NCs and Ag NCs, analytical applications of the copper nanoclusters (Cu NCs) are relatively limited and still at an early stage. In this review, we focus on recent advances in the analytical applications of Cu NCs based on their optical, electrochemical, and catalytical properties for the detection of various analytes, including metal ions, anions, biomoleculars (proteins, nucleic acids etc.), small molecules and pH. In addition, their applications in biological labeling and bioimaging were summarized. Keywords: Copper nanoclusters Fluorescent detection Chemiluminescent detection Colorimetric detection Electrochemical detection Bioimaging Biolabeling Fluorescent probe Sensing Metal ions 

Corresponding author.

E-mail address: [email protected] (W. Fan); [email protected] (Y. Huang)

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1. Introduction Metal nanoclusters (MNCs) are composed of very few atoms, with a core size in the sub-nanometer regime, providing the missing link between atomic and nanoparticle behavior in metals [1]. Because their sizes are comparable to the Fermi wavelength of electrons, which results in molecule-like properties including discrete electronic states and size-dependent fluorescence (FL) [2]. Exploiting these unique properties has resulted in many important findings of MNCs, and MNCs have drawn considerable research interest in recent year years due to their unique electrical, physical and optical properties for use in electronic devices, catalysis, biological imaging, and chemical sensors [3-6]. In particular, compared to quantum dots and organic dyes, MNCs exhibit excellent photostability, large Stokes shift, and low environmental hazard [7,8]. Inspired by the existing and potential applications of MNCs, studies on MNCs are growing rapidly in the past few years. To date, great efforts have been paid to noble metal nanoclusters (mainly Au and Ag) due to their chemical stability and facile synthetic procedure [9-11]. However, very promising copper nanoclusters (Cu NCs) have received less attention. In fact, compared to the noble metal Au and Ag, the metal Cu is significantly cheaper and widely used in industries because of its high conductivity, similar properties to Au and Ag. Furthermore, compared with the expensive precursors for the synthesis of Au NCs and Ag NCs, the precursor for the preparation of Cu NCs is relatively abundant, inexpensive and readily available from commercial sources, therefore the Cu NCs are more favorable for various applications than the noble metal NCs. In this review, we mainly focus on the analytical applications of Cu NCs in the past few years rather than their synthetic methods, which have been reviewed in detail in previous publications [5,10,11]. However, for every analytical application, we will briefly introduce the synthetic strategy for the Cu NCs and then their analytical applications. In the coming text, we highlight recent advances in the analytical applications of the Cu NCs-based optical, electrochemical, and catalytical methods for the detection of various analytes, including metal ions, anions, biomolecular (proteins, nucleic acids etc.), small molecules and pH. Also, their applications in biological 2 Page 2 of 30

labeling and bioimaging were covered. And these recent applications are summarized in Table 1. 2. Photophysical properties of Cu NCs When metal nanoparticles (NPs) change to metal NCs, the properties of particles disappear and the bands turn into more or less discrete energy levels. Thus, the collective oscillation of electrons is obstructed and metal NCs do not give rise to surface plasmon resonance effect, and exhibit unique optical properties such as molecule-like absorption and strong luminescence [5]. It is well known that the UV-Vis absorption of Cu NPs is dominated by surface plasmon resonance peaks at 560600 nm [12]. However, significantly different from large Cu NPs, the UV-Vis absorption of Cu NCs exhibit molecular-like optical transitions with absorbance bands between 216 nm and 468 nm (Table 1). These molecular-like optical transitions in UV-vis absorption spectrum are due to the quasi-continuous electronic energy band structure and quantum confinement effects of Cu NCs. Similar to that of Au NCs and Ag NCs, the fluorescence properties of Cu NCs are dependent on their chemical environments, including the cluster size, solvent and surface capping ligands or Cu NCs prepared from different synthetic strategies etc (Table 1). 3. Cu NCs-based fluorescence sensors The fluorescent Cu NCs always have been applied as optical probes for the detection of various targets such as metal ions, anions, biomoleculars, small molecules and solution pH. It is also an attractive choice for the biological labeling and imaging owing to their unique features, like optical activity, biocompatibility, and nontoxicity. In the following section, we highlight recent advances in the analytical applications of Cu NCs based on their fluorescent properties. 3.1. Metal ions sensors The Cu NCs have been used as new fluorescent probes for the detection of heavy metal ions, including Hg2+, Pb2+, Cu2+, Fe3+, Cr(VI) and Cr3+. 3.1.1 Hg2+ sensor Ghosh et al. [13] found that hydrazine reduction of Cu2+ derived Cu NCs stabilized by citric acid and CTAB can be used as a fluorescence probe to detect Hg2+ 3 Page 3 of 30

based on its quenching effect of the fluorescent Cu NCs, with a limit of detection (LOD) of 1 nM. Also, Yang et al. [14] synthesized L-cysteine Cu NCs, which were used for assaying Hg2+ based on the FL quenching of the Cu NCs due to strong interactions between Hg2+ and L-cysteine on the surface of the Cu NCs. The linear detection range for Hg2+ is 0.1 – 1000 µM with a LOD of 24 nM. In contrast to previously reported preparation of fluorescent Cu NCs using ds-DNA as templates, Liu et al. [15] used ss-DNAs as templates to prepare Cu NCs in the presence of Hg2+. It is found that upon addition of Hg2+, a more intense fluorescence response was observed. This is caused by Hg2+ mediated T–T base pair to favor the formation fluorescent Cu NCs, demonstrating the key role of DNA thymine in producing red-emissive fluorescent Cu NCs on ss-DNA templates. Due to highly specific role of Hg2+ in mediating a T–T base pair, a highly selective turn-on fluorescence sensor was designed for the detection of Hg2+. Using 34T as templates, the limit detection of 10 nM is obtained, which is comparable to the permitted maximum level (about 10 nM) for Hg2+ in drinking water from the United States Environmental Protection Agency (EPA). Recently, Liao et al. [16] reported a new strategy for the fast synthesis of Cu NCs by using BSA and hydrogen peroxide, in which H2O2 plays both a ligand to combine with BSA–Cu complex to form BSA–Cu–H2O2 complex and an oxidizing agent to partly destroy disulfide bonds in BSA. The obtained Cu NCs showed strong fluorescence emission at 420 nm. Interestingly, it was found that Hg2+ can significantly quench the FL of as-prepared Cu NCs through introduction of Cu NCs aggregation due to formation of Hg–S covalent bond and a part destruction of Cu–S bonds in Cu NCs by Hg2+. The as-prepared Cu NCs provided ultrahigh sensitivity for the detection of Hg2+ with a LOD of 4.7 pM. 3.1.2 Pb2+ sensor Early in 2011, Goswami et al. [17] succeeded in BSA templated synthesis of fluorescent Cu NCs from CuSO4 precursor under alkali media for the first time and subsequently used for selective sensing of Pb2+ at the part-per-million level. The detection mechanism is based on the luminescence quenching of the Cu NCs in the 4 Page 4 of 30

presence of Pb2+ due to the Cu NCs aggregation induced by the complexation between BSA and Pb2+ ions. Based on a similar principle, in another work, Chen et al. [18] introduced the use of dsDNA as template for the preparation of Cu NCs through Cu2+ reduction by ascorbate according to the previously reported method [19,20]. The Stern–Volmer plot of the fluorescence quenching by Cu2+ displayed a linear range of 5 to 100 nM with a LOD of 5 nM, which is much lower than the maximum permitted level (72 nM) of Pb2+ in drinking water by EPA. Another interesting piece of work introduced the use of the electrochemically synthesized Cu NCs as FL probe for the detection of Pb2+ ions [21]. In this work, Vilar-Vidal et al. found that medium copper (Cu13) clusters are very selective for Pb2+ ions by FL quenching and exhibited a LOD of 4.9 M. However, small Cu NCs (Cu7) show no selectivity and large ones (Cu20) are not sensitive to the ions. Additionally, they found that the as-synthesized Cu NCs display highly photocatalytic activity, thus can be used for the photocatalytic elimination of Pb2+ ions. Such effects are attributed to an efficient electron transfer due to the LUMO (Lowest Unoccupied Molecular Orbital) energy of the cluster overlapping with the ion redox potential, which can be used for the explanation of previously reported results for different types of metal clusters. Recently, Wang et al. [22] reported a new Cu NCs-based method for Pb2+, in which the glutathione (GSH)-Cu NCs were easily synthesized via one-pot sonochemical route, using GSH both as stabilizing agents and reducing. The obtained GSH-Cu NCs exhibited red fluorescence emission at 606 nm with a QY of 5.3% and their luminescent properties show pH dependent. It was found that Pb2+ cause significant fluorescence quenching of GSH-Cu NCs by introducing Cu NCs aggregation due to high affinity of Pb2+ to GSH on the surface of Cu NCs. The linear range and limit of detection for Pb2+ are 1 to 160 nM and 1 nM, respectively. Additionally, the GSH–Cu NCs show low toxicity and good biocompatibility, and have been successfully used for monitoring Pb2+ in living cells. 3.1.3 Cu2+ sensor Zhong et al. [23] found that the fluorescence of the BSA coated Cu NCs can be 5 Page 5 of 30

quenched by Cu2+ through the formation of the BSA-Cu NCs aggregates due to the paramagnetic behavior of Cu2+. Interestingly, Cu2+ is removed from the surface of BSA-Cu NCs upon addition of EDTA due to the higher chelating ability of EDTA for Cu2+. On this basis, a reversible fluorescence sensor was developed for the detection of Cu2+ in aqueous media. The linear detection range for Cu2+ is 0.02 to 34 μM with a LOD of 1 nM. Li et al. [24] developed a simple method for the detection of Cu2+ by a FL turn-on strategy, which is based on the formation of red fluorescent Cu NCs with D-Penicillamine in ethanol media through solvent induced formation mechanism. By using ethanol to replace water as reaction media, the fluorescent Cu NCs were formed very quickly. The Cu2+ could be selectively detected within minutes with a LOD of 0.3 ppm. Gui et al. [25] proposed a facile photoreduction approach for the synthesis of Cu NCs in the presence of multidentate polymers {poly(acrylic acid)-graftmercaptoethylamine, PAA-g-MEA} as stabilizing agents without the need of additional reductive. The resulting Cu NCs exhibited bright red fluorescence emission at 630 nm with a QY of 5.7%. It was found that ammonium pyrrolidine dithiocarbamate (APDC) caused quenching of Cu NCs emission through Cu–APDC complex formation, resulting in partial loss of the surface Cu-thiol layers. However, addition of Cu2+ leads to recovery of FL emission of Cu NCs due to formation of Cu–thiol complex again. Hence, an “off-to-on” fluorescence probe could be designed for selective Cu2+ sensing. 3.1.4 Fe3+ sensor Feng et al. [26] synthesized branched polyethyleneimine (BPEI) capped Cu NCs, in which BPEI served as templates and ascorbic acid acted as reductant. The resulting Cu NCs show luminescence properties having excitation and emission maxima at 360 nm and 430 nm, respectively. They also promise good water-solubility, photostability and high stability even in high salt media. It is found that Fe3+ caused significant fluorescence quenching of the BPEI-Cu NCs via electron transfer, while other commonly existing cations and anions had minor effect on the fluorescence intensity 6 Page 6 of 30

of the BPEI-Cu NCs. On this basis, a sensitive and selective fluorescence sensor for detection of Fe3+ ions was established and applied to the detection of Fe3+ in water and human urine samples. The limit of detection is 340 nM and linear detection range is 0.5–1000 M. Recently, our groups [27] have designed the water soluble fluorescent Cu NCs using small molecule tannic acid (TA) as ligands and ascorbic acid as reducing in a one-step facile route. The as-prepared Cu NCs exhibit the fluorescent excitation and emission maxima at 360 nm and 430 nm, respectively, with a quantum yield of about 14%. The TA-Cu NCs are very stable and their luminescent properties show pH independent. It is found that Fe3+ caused a strong FL quenching of the TA-Cu NCs due to the fact that Fe3+ easily combined with the surface of Cu NCs through complex formation with tannic acid of the TA-Cu NCs via an electron transfer mechanism. We have also demonstrated that the quenching abilities caused by Fe3+ decreased with increasing the size of the prepared TA-Cu NCs. Based on above finding, a facile chemosensor was developed for sensing of Fe3+ ions with detection limit as low as 10 nM and a dynamic range from 0.01 to 10 M. TA-Cu NCs show a high selectivity for sensing of Fe3+ ions, other common metal-ions, including alkali, alkaline earth, and transitional metal ions have no obvious effects on the FL emission of Cu NCs. The high selectivity was attributed to the strong electron-accepting ability of Fe3+ because the outer electronic structure of Fe3+ is 3d54s0, and the five d orbits are half-filled, resulting in its relatively high charge density with the stronger electron-withdrawing ability. Successful application for the detection of iron contents in serum samples was demonstrated. 3.1.5 Chromium sensor Chromium, in particular Cr(VI), was recognized as toxic heavy metals. Cui et al. [28] used cysteine stabilized Cu NCs as a fluorescent probe for the determination of Cr(VI). They synthesized Cu NCs by reduction of Cu(II) in the presence of cysteine as reductant and stabilizer under strong alkali media (pH 12). The obtained Cu NCs show green fluorescence with the maximum of emission at 490 nm and a quantum yield of 5.6 %. It was found that Cr(VI) showed efficient quenching of green fluorescence of the Cu NCs, providing a simple and sensitive fluorescent assay to 7 Page 7 of 30

detect Cr(VI). The linear detection range is 0.2 to 60 μM with a LOD of 60 nM. Singh et al [29] synthesized serial heterotripodal receptors with potential cation binding sites, which could be transformed into organic nanoparticles (ONPs) in aqueous due to the presence of extensive intermolecular and intermolecular hydrogen bonding. The formed ONPs can be used as ligands to prepare Cu NCs due to their peculiar ability for recognition of Cu2+, leading to reduction of Cu2+ to Cu(0) and formation of ONPs@CuNCs hybrid. The ONPs@CuNCs hybrid exhibited red fluorescence with maximum emission peaks at 620 nm upon excitation at 468 nm. Upon addition of Cr3+ ions, the agglomerization of the Cu NCs occurred because of adsorption of Cr3+ ions on the surface of Cu NCs due to electrostatic interactions, resulting in fluorescence quenching of the Cu NCs. Thus, ONPs@CuNCs hybrid can be used as fluorescent probes for detection of Cr3+ in aqueous phase with a LOD of 3 μM. 3.2. Anions sensors Recently, several investigators have reported the potential of Cu NCs as fluorescent probe for sensing of anions. These include cyanide (CN), I, phosphate ion and hypochlorite. Lin et al. [30] synthesized Cu NCs from copper nitrate and mercaptobenzoic acid (MBA) as reducing and capping agent. They investigated the effect of different isomers of MBA on the fluorescence properties of the resulting Cu NCs. Interestingly, it was found that thiosalicylic acid (TA)–Cu NCs exhibited blue emission at 420 nm and a QY of 13%, however, 3-MBA-CuNCs and 4-MBA-CuNCs showed red fluorescence emission at 668 nm and 646 nm, respectively, and low QYs of 0.04% and 0.5%, respectively. In addition, 4-MBA–Cu NCs show an interesting aggregation-induced emission (AIE) effect, while TA–Cu NCs and 4-MBA–Cu NCs display different pH-dependent fluorescence properties upon increasing pH values. Importantly, CN can act as a selective quencher of the TA–Cu NC as a result of Cu(CN)43 formation at a high concentration above 1 M or complex formation between Cu2+ and CN at a low concentration below1 M. On this basis, a new fluorescent probe was designed for the selective detection of CN with a LOD of 5 nM, which is 540-fold lower than the maximum contaminant level (2.7 M) in 8 Page 8 of 30

drinking water permitted by WHO. Zhong et al. [31] prepared polyethyleneimine (PEI) protected Cu NCs as novel fluorescent probes for the detection of I. The fluorescence of PEI-Cu NCs was quenched in the presence of I ions; other anions have minor effect on fluorescence response of PEI-Cu NCs. A detection limit of 100 nM was obtained for I. The method was successfully used for the detection of I− in urine samples. Our group [32] reported an off to on fluorescence probe for sensitive and selective phosphate (Pi) sensing, which was based on the competition between Pi and tannic acid stabilized copper nanoclusters (TA–Cu NCs) for Eu3+ binding. Eu3+ can quench the FL of Cu NCs through static quenching mechanism by acting as a bridge for the induction of Cu NCs aggregation, and then are removed from the surface of Cu NCs after Pi introduction due to the strong interactions between Pi and Eu3+, thus the FL turns on. The Cu NCs provided excellent selectivity for Pi over other metal ions and anions. The limit of detection for Pi was calculated to be 9.6 nM. In another work, we synthesized PVP capped Cu NCs using formaldehyde as reducing agent for the detection of ClO [33]. The PVP-Cu NCs exhibit an excitation-dependent fluorescent property, and promise long-term storage stability (at least 2 months) and high concentration of salt tolerance. They displayed a strong blue emission at 430 nm with a quantum yield of about 13%. The hypochlorite causes significant fluorescence quenching of the PVP-Cu NCs, while other common cations, anions and hydrogen peroxide have minor effects on the fluorescence. The linear detection range for hypochlorite is 1 to 30 µM with a LOD of 0.1 µM. Recently, Li et al. [34] reported a novel, turn-on fluorescent assay for S2− using cysteine-capped Cu NCs as a fluorescent probe. The luminescent Cu NCs were prepared via the process of size-focusing etching from nonluminescent nanocrystals using cysteine as a model ligand. The detection mechanism was based on the fluorescence enhancement of cysteine-capped Cu NCs by S2− upon interaction with the surface of the Cu NC via the Cu–S bonds. It is found that upon addition of S2−, a more intense fluorescence response was observed. This was caused by S2− mediated aggregation of the Cu NCs, which was similar to solvent-induced aggregation. The 9 Page 9 of 30

linear detection range for S2− is 0.2–50 M with a LOD of 42 nM. Furthermore, they observed that S2−-induced fluorescence enhancement of cysteine-capped Cu NCs occurred in aqueous media without the need of organic solvent, thus providing a novel strategy to use the AIE properties of the Cu NCs for sensing other analytes in aqueous media. 3.3. Small molecule sensors Some researchers have reported sensing of small molecules analytes by fluorescence quenching of Cu NCs. Zhou et al. [20] illustrated the successful application of Cu NCs as a label-free fluorescent aptamer sensor for the detection of ATP with high sensitivity and selectivity. The principle of ATP sensing is based on quenching the fluorescence of dsDNA-templated Cu NCs. Under optimal conditions, the linear detection range for ATP is 0.05500 μM with a LOD of 28 nM. Note that the proposed method promises high selectivity for ATP detection due to specific interactions between the nucleic acid aptamer and ATP. Furthermore, it is simple without the complicated design of the fluorescent aptasensors and use of labeled aptamer, and can be easily extended to a label-free detection of other small molecules analytes, such as cocaine. Hu et al. [35] reported a fluorescence turn-off strategy for biothiols, including glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The detection mechanism is based on the fluorescence quenching of the Cu NCs by biothiols, which is likely caused by the formation of a coordination complex by the Cu–S metal–ligand bond between the dsDNA-CuNCs and the biothiols. The Cu NCs displayed a strong emission band at 580 nm and exhibited a linear response to GSH, Cys and Hcy in the range 2.0×10-6 to 8.0×10-5 M, 2.0×10-6 to 1.0×10-4 M and 5.0×10-6 to 2.0×10-4 M, respectively. The limits of detection for GSH, Cys and Hcy were calculated to be 2, 2, 5 μM, respectively. Deng et al. [36] found that blue emission of BSA templated Cu NCs is quenched by 2,4,6-trinitrophenol (TNP) due to Förster resonance energy transfer (FRET) from fluorescent Cu NCs to TNP. The method was favorable for the detection of TNP in the range 0.8 to 100 μM with a LOD of 120 nM. Another example was reported in the 10 Page 10 of 30

sensing of Sudan I-IV dyes in food samples, which is based on FL quenching of PEI-capped Cu NCs emission by Sudan dyes through inner filter effect [37]. Ma et al. [38] reported on the use of an penicillamine (PA)-Cu NC for sensing H2S, which can cause photoluminescence quenching of PA-Cu NC through the formation of CuS NPs. Interestingly, properties of PA-Cu NCs largely depended on solvent molecular, particularly in the case of DMF and DMSO. In 65% DMF, the PA-Cu NC aggregates display good stability, showing strong yellow emission at 580 nm. The probe allowed the detection of H2S with a linear range of 1–100 μM and a LOD of 500 nM. Lin et al. [39] reported on the use of PEI-Cu NCs for the quenching-based sensing of hydrogen peroxide due to H2O2 induced oxidation of Cu NCs. When combined with glucose oxidase, PEI-Cu NCs was proposed for the sensing of glucose in serum samples. A similar example was reported in the sensing of hydrogen peroxide and glucose by D-penicillamine (DPA) templated Cu NCs [40]. The linear range for H2O2 is 0.05 to 2 mM with a LOD of 0.01 mM. Zhou et al. [41] synthesized dihydrolipoic acid stabilized Cu NCs for sensing of hydrogen peroxide. The detection mechanism is based on the fluorescence quenching of the Cu NCs by H2O2, which is likely caused by the oxidation of the metal core. The linear range for H2O2 is 1 to 10 µM, deriving a LOD 0.3 µM. The method was applied successfully to the detection of H2O2 in human urine samples. Recently, Miao et al. [42] prepared papainfunctionalized Cu NCs for the detection of H2O2. It was found that the as-prepared Cu NCs displayed red fluorescence at 620 nm and a QY of 14.3%. Addition of H2O2 induced FL quenching of the as-prepared Cu NCs, which was caused by the conversion of H2O2 to OH in the presence of the as-prepared Cu NCs. On this basis, H2O2 was determined in the linear range of 1 to 50 µM with a LOD of 0.2 µM. The method was applied successfully to the detection of H2O2 in lake water samples. Gao et al. [43] reported BSA-capped Cu NCs could be used as a FL probe capable of recognizing kojic acid (KA) through static quenching due to the non-fluorescent ground-state complex formation between KA and Cu(II) on the surface of Cu NCs. Due to specific interaction between KA and cooper ions, the 11 Page 11 of 30

proposed method was highly selective for KA. The probe was favorable for the detection of KA in the range 0.2 to 50 μM with a LOD of 70 nM. Zhao et al. [44] reported a fluorescent probe for guanosine 5’-triphosphate (GTP) sensing by Cu NCs, which was prepared by reduction of CuCl2 in the presence of histidine acting as both reductive and capping agent. It was found that blue emission of the Cu NCs can be effectively quenched by GTP. However, other nucleoside triphosphates, such as ATP, CTP and UTP, have minor quenching effect, showing the good selectivity of the Cu NCs for GTP detection. Zhu et al. [45] reported a new FL method for the detection of melamine by using poly-thymine (T) stabilized Cu NCs as a fluorescence probe, which was prepared by using CuSO4 as precursor and ascorbate as reducing agent in the presence of poly T as capping agent. The poly-T stabilized Cu NCs show red fluorescence at 598 nm. Interestingly, in the presence of melamine, the fluorescence intensity of poly-T stabilized Cu NCs was obviously enhanced. This is caused by hydrogen bond formation between thymine and melamine, resulting in formation of double strand poly T–melamine complex with a more rigid structure. Due to specific and strong interaction between melamine and thymine bases, a highly selective turn-on fluorescence sensor was designed for the detection of melamine. The linear detection range for melamine is 0.1 to 6 µM. The limit detection of 95 nM is obtained, which is 200-fold lower than the US Food and Drug Administration estimate melamine safety limit of 20 M. The method was applied successfully to the detection of melamine in milk samples. 3.4. Protein detection Based on inhibition effect of pyrophosphate (PPi) on the dsDNA-templated formation of FL Cu NCs, Zhang et al. [46] proposed a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions by using PPi as its substrate. Due to strong interaction between PPi and Cu2+, dsDNA-templated Cu NCs formation was greatly inhibited, thus leading to a low fluorescence. However, with the introduction of ALP, PPi was hydrolyzed to Pi, this disable the complexation between Cu2+ and PPi, which favor the formation of FL 12 Page 12 of 30

Cu NCs, thus the FL turns on. A linear detection range for ALP is 0.1 to 2.5 nM, with a LOD of 0.1 nM. In another work, Zhang et al. [47] developed a simple, sensitive, low-cost and label-free method to detect 3’–5’ exonuclease activity. They used dsDNA as templates for Cu NCs formation and substrate of the 3’–5’ exonuclease. The resulting Cu NCs showed strong red fluorescence at 575 nm. However, in the presence of the ExoIII, such a FL emission decreased significantly because the stable DNA duplexes will be cleaved into fragments due to the high exodeoxyribonuclease activity of ExoIII. This was applied for the detection of Exo III activity. The method exhibited a low detection limit of 0.02 U mL-1 for Exo III, and there was a good linear correlation between the fluorescence intensity and the concentration of Exo III in the range of 0.05 to 2 U mL-1. 3.5. Nucleic acid detection DNA oligonucleotides have been used as template to prepare the Cu NCs, which make it possible to detect nucleic acids. The first example of using Cu NCs for the potential detection of DNA was reported by Rotaru et al.[19]. They synthesized Cu NCs with various DNA structures, and found that the dsDNA is more selective than the ssDNA for the formation of Cu NCs. The resulting Cu NCs showed strong fluorescence at 587-600 nm. Additionally, they found that the size of the Cu NCs formed can be easily regulated by the length of dsDNA template. The longer the length of dsDNA template, the larger the particle size of the formed Cu NCs. Jia et al. [48] found that fluorescent DNA-hosted Cu NCs is very sensitive to base type located in the major groove of DNA, making it possible to identify single-nucleotide polymorphisms. Interestingly, they found that the fluorescence of DNA-hosted Cu nanoclusters displays mismatch dependent (Fig. 1), thus the Cu NCs might become a simple yet effective probe for detecting not only a single mismatch but also its mismatch type in a specific DNA sequence. Wang et al. [49] developed a dsDNA-Cu NCs-based label-free fluorescent method for sensitive and selective detection of miRNAs, which was based on the formation of fluorescent Cu NCs derived from the DNA duplex templates induced by hybridization between DNA and target miRNA. The resulting Cu NCs showed strong fluorescence at 608 nm. In order to obtain high sensitivity, the target-triggered 13 Page 13 of 30

isothermal exponential amplification reaction was used. The linear detection range for miRNAs is 1 pM to 10 nM with a LOD of 1 pM. In addition, the method promises high sequence specificity, thus can be used for discriminating the difference between miRNA family members. Xu et al. [50] designed a novel strategy for the detection of microRNA based on the formation of concatemeric dsDNA-templated Cu NCs by the rolling circle replication (RCR) technology. In this strategy, the PCR process was triggered by a primer in presence of phi29 polymerase, dNTPs, and the circular template, which would form a long concatemeric ssDNA. The formed ssDNA hybridized with microRNA to form a long concatemeric dsDNA acting as templates to synthesize Cu NPs.

Compared

with

monomeric

dsDNA-Cu

NCs,

the

concatemeric

dsDNA-templated Cu NCs promised significantly enhanced stability and very high sensitivity for the target with about 10000-fold amplification. Therefore, it could selectively and sensitively detect microRNA with a linear range from 10 to 400 pM and a LOD of 10 pM. Most recently, Chen et al. [51] demonstrated a new and real application of label-free fluorescent Cu NCs for genotyping of deletion and duplication of Duchenne muscular dystrophy (DMD) in terms of different fluorescent intensities. By using the exons of DMD gene as the model analytes, they found that after PCR reaction, the deletion type does not show fluorescence because it could not form dsDNA amplicones, thus does not support the formation of Cu NCs; however, the duplication type emits higher fluorescence than normal type. This can be used to discriminate deletion or duplication genotypes of DMD in a real clinical sample with the help of PCR reaction. The proposed method has proved to be successful in the detection of deletion and duplication types in DMD-dominated exons 45, 46 and 47 in real clinical samples, and the results agree well with those obtained by multiplex ligation dependent probe amplification (MLPA). 3.6. pH sensor The successful use of fluorescent Cu NCs as pH sensor was first reported in 2014 by Wang et al. [52]. They synthesized BSA-Cu NCs using hydrazine hydrate as a reducing agent, and the resulting BSA-Cu NCs showed red fluorescence emission at

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620 nm with quantum yield of 4.1%. It was found that when the pH varied from 12 to 6, the fluorescence response of the BSA-Cu NCs increased by about 2 times, and showed a good reversibility between pH 6 and 12. Interestingly, the fluorescence response peaked at nearly neutral pH range from 6 to 7, suggesting that the obtained BSA–Cu NCs were very suitable for cellular imaging. More recently, by using GSH as a reducing agent and a capping agent, they successfully prepared pH-responsive GSH–Cu NCs via a one-pot sonochemical reaction [22]. The resultant GSH-Cu NCs displayed a red fluorescence emission at 606 nm with a QY of 5.3%. In the pH range of 4–9, the GSH–Cu NCs showed fast and reversible responses to pH values. In order to extend the pH response range, Wang et al. [53] reported a pH sensor based on trypsin stabilized fluorescent Cu NCs. It was found that the fluorescence response of the trypsin-stabilized Cu NCs decreased linearly and reversibly with increase in solution pH from 2.02 to 12.14. Similarly, Liao et al. [54] designed a novel BSA-capped Cu NCs for sensitive and wide-range pH sensing in range of 2–14. The BSA-Cu NCs were prepared by using hydrogen peroxide as additive to partially destroy the peptide and disulfide bonds in the BSA molecule, leading to the exposure of hydrophilic groups capable of protonation. It is noted that although wide pH response range was realized in above two works, the fluorescence emission of the sensors shifted to blue region, which might limit their application in biological samples. 3.7. Biological labeling and imaging In the past few years, luminescent Cu NCs have been increasingly investigated for biolabeling and bioimaging applications due to their ultrafine size, good biocompatibility and nontoxicity. In our group, fluorescent Cu NCs capped with tannic acid have been successfully synthesized at mild conditions [27]. CCK-8 assays have proved very low cytotoxicity of the as-synthesized Cu NCs and good biocompatibility. The as-prepared Cu NCs have been successfully used for imaging Fe3+ in living cells. Ghosh et al. [55] reported a one-step method for the synthesis of lysozyme stabilized Cu NCs, which showed strong bright-blue fluorescence emission peak at 450 nm when excited by 360 nm light and a quantum yield of 18%. They were 15 Page 15 of 30

successfully used for labeling of the cervical cancer HeLa cells, which could easily be observed under UV light (340−380 nm). Wang et al. [52] found that the fluorescence response of the BSA-Cu NCs peaked at nearly neutral pH range from 6 to 7. Thus, the obtained BSA–Cu NCs were very suitable for cellular imaging. Huang et al. [56] reported the synthesis of fluorescent Cu NCs with diameters of 1.70.4 nm by a peptide with amino acid sequence CLEDNN as a template. The synthesized Cu NCs displayed an intense blue fluorescence at 454 nm and good stability with low cytotoxicity, thus had been successfully used as a fluorescent probe for imaging HeLa cells. Interestingly, their fluorescence response shows temperature dependent. When temperature increased from 10 to 55 C, their fluorescence intensity decreased linearly, showing the high potential of using the Cu NCs as a fuorescent probe for temperature sensing. Zhao et al. [57] reported an interesting use of the transferrin (Trf)-functionalized Cu NCs (Trf-Cu NCs) as a novel red-emitting fluorescent probe for the targeted bioimaging of cancer cells. The Trf-Cu NCs were prepared by a simple one-pot procedure through reduction of CuSO4 with ascorbic acid in the presence of Trf, which acted as a stabilizer, a reducer, and a functional ligand for targeting the transferrin receptor. The prepared Trf-Cu NCs displayed an intense red fluorescence with an emission peak at 670 nm and a quantum yield of about 6.2%. Importantly, their cytotoxicity was negligible because Trf could prevent any leakage of Cu effectively. Due to highly specific affinity of Trf to transferrin receptor (TfR), the probe was successfully used to the targeted bioimaging of HeLa cells (with a high level of expression of TfR) within a shorter incubation time. Das et al. [58] showed the application of ultrafine Cu NCs with blue emission in tumor cell imaging. The Cu NCs were synthesized from copper nitrate by using glutathione both as a protecting group and reducing agent. The as-synthesized Cu NCs exhibited intense blue fluorescence maximized at 430 nm, excellent photostability, superior biocompatibility and no toxicity. The cell-imaging results demonstrate that the Cu NCs localize primarily in nuclear membranes of the different cancerous (Hela, MDAMB-231, and A549) cells, showing great potential to be used 16 Page 16 of 30

as a nontoxic and biomedical luminescent probe. Interestingly, upon addition of Fe3+ ions, the electron transfer between as-synthesized Cu NCs and Fe3+ ions occurred, resulting in fluorescence quenching of the Cu NCs. Thus, the as-synthesized Cu NCs can be used as fluorescent probes for detection of Fe3+ in aqueous phase with a detection limit of 25 nM. Recently, Gao et al. [59] reported an interesting use of the chelator free radioactive [64Cu]Cu nanoclusters ([64Cu]CuNC@BSA) for positron emission tomography (PET) imaging. The [64Cu]CuNC@ BSA was facially prepared by a simple one-pot chemical reduction strategy by using BSA as a template, in which 64Cu is an integral building block of CuNC@BSA rather than chelated to the nanoclusters. As compared to radiometal-chelator complexes-based PET imaging agent, the [64Cu]CuNC@ BSA exhibited high radiolabeling stability, ultrasmall size, and rapid deposition and diffusion into tumor, as well as predominantly renal clearance. Additionally, through the conjugation of BSA to the tumor target peptide luteinizing hormone releasing hormone (LHRH), the [64Cu]CuNC@BSA-LHRH was obtained. The LHRH in [64Cu]CuNC@BSA-LHRH plays a functional ligand for targeting the LHRH receptors, which are overexpressed in some cancer cells. It was found that [64Cu]CuNC@BSA-LHRH showed 4-flod higher tumor uptake than that of [64Cu]CuNC@BSA, showing the high potential as a tumor PET imaging agent for early diagnosis of cancer. Compared with near-infrared fluorescence imaging, the as-prepared Cu NCs as tracers showed more sensitive, accurate, and deep penetration imaging of orthotopic lung cancer in vivo, which favored their potential in PET molecular imaging. More recently, Zhu et al. [60] reported a green staining method of DNA in polyacrylamide gel electrophoresis through in situ synthesis of DNA-templated fluorescent Cu NCs in the gel by successively and repeatedly (twice) immersing polyacrylamide gel into buffered CuSO4 solution (pH 7.5) and ascorbic acid. The synthesized Cu NCs exhibited an orange-red fluorescence with an emission peak at 584 nm when excited at 343 nm. Thus the DNA bands in the gel can be visible under UV light irradiation. Importantly, the result of skin toxicity tests using rat suggests 17 Page 17 of 30

that the Cu NCs had no potential toxicity in contact with skin, indicating that the CuNCs staining is a green method. 4. Cu NCs-based chemiluminescence assay Chen et al. [61] reported a new amplified chemiluminescence in the NaHCO3-H2O2 system mediated by the zinc-copper bimetal NCs as catalyst. They synthesized the bimetal NCs by a simple one-pot method by using BSA as a template. The synthesized Zn/Cu@BSA NCs under optimized conditions showed the extensive blue emission at 430 nm. Upon addition of Zn/Cu@BSA NCs into NaHCO3-H2O2 system, a remarkable CL enhancement was observed. In terms of the ESR results, UV-vis absorption, fluorescence, and CL spectra of the NaHCO3-Zn/Cu@BSA NC-H2O2 system, it is suggested that hydrogen peroxide reacts with NaHCO3 to form peroxymonocarbonate (HCO4) during CL reaction. As shown in Fig. 2, the Zn/Cu@BSA NCs catalyzed decomposition of HCO4 to generate key radicals including OH and CO3, accompanied by the structure change of BSA protein. This resulted in the aggregation of metal nanoclusters to metal nanoparticles due to loss of protective BAS scaffold. Simultaneously, OH reacts with excess HCO3 to form excited (CO2)2, the coupling of excited (CO2)2 to surface plasmons of Zn/Cu NPs further enhanced the CL. As a result, aggregation-induced structural change of protein-stabilized zinc/copper NCs results in amplified chemiluminescence. On this basis, a new CL method was developed for the sensitive and selective detection of H2O2 in environmental samples. The linear detection range for H2O2 is 0.005 to 1 M with a LOD of 0.3 nM. Xu et al. [62,63] reported the use of Cu NCs in traditional luminal CL reaction. In their first work [62], a 70-fold CL enhancement was found in H2O2luminol system by the BAS templated Cu NCs as catalyst. Additionally, some organic compounds containing OH, NH2, SH groups could inhibit the CL response of luminol–H2O2–Cu NCs system, making it possible to use the luminol–H2O2–Cu NCs for the detection of these compounds. As an example, the proposed method was applied to H2O2 detection. Under optimum conditions, the CL intensity displayed a calibration response for H2O2 over 3 orders of magnitude from 0.1 to 150 mM with a 18 Page 18 of 30

LOD of 0.03 mM. In another work [63], based on inhibition effect of bisphenol A (BPA) on Cu NCs enhanced luminol–KMnO4 CL system, a highly sensitive CL method was developed for the determination of BPA in aqueous samples. The detection for BPA is 0.12 nM. 5. Cu NCs-based electrochemical sensors Previous studies have shown that nanometer-sized copper clusters exhibit high catalytic and electrocatalytic activities compared to the inert bulk copper metal. On this basis, Dai et al. [64] proposed a novel electrochemical biosensor for miRNA detection by adopting DNA-miRNA heteroduplexes as novel templates to form Cu NCs. The formed Cu NCs could electrocatalyze H2O2 reduction, generating steady and amplified electrochemical signals, which could be used to determine miRNA. The linear detection range for miRNA is 25 to 300 fM, deriving a LOD of 8.2 fM. The biosensor proved to be selective and used repeatedly in determining single nucleotide polymorphism. More importantly, this approach could be versatile by simply changing the corresponding DNA simply. Xia et al. [65] developed a micro electrochemical sensor for nitrate and total nitrogen via electrodepositing copper onto the working-electrode by cyclic voltammetry (CV) method and square-wave pulsating current (PC) method, which are based on electrochemical reduction of nitrate catalyzed by Cu NCs. The copper layer fabricated by PC displayed higher sensitivity for nitrate detection than that by CV method, due to the open porous structure of the prepared copper layer. For TN detection, the microsensors modified by PC method had sensitivity of 7.3104 μA/(mg L1) and a LOD of 0.1 mg/L for TN determination. Kang et al. [66] constructed a nonenzymatic glucose biosensor via electrodepositing Cu nanoclusters on the multiwall carbon nanotube (CNTs)-modified GCE. The nonenzymatic sensor revealed synergistic electrocatalytic activity to the oxidation of glucose in alkaline media. The proposed glucose biosensor shows a good linearity between the current response and glucose within a range from 7.0×10-7 to 3.5×10-3 M with a LOD of 2.1×10-7 M. It was successfully applied for the detection of glucose in real blood serum samples. 19 Page 19 of 30

6. Cu NCs-based colorimetric sensors Cu NCs-based colorimetric methods are based on their enzyme-like property. For example, Hu et al. [67] synthesized water-soluble Cu NCs by using bovine serum albumin (BSA) as a stabilizer and reducer. As shown in Fig. 3, the Cu NCs exhibit peroxidase-like activity and can catalyze the oxidation of TMB by H2O2 to produce a blue color reaction, which can be applied to detect H2O2 with a linear range from 0.01 to 1 mM and a LOD of 10 μM. When coupled with GOx, Cu NCs can be used to detect glucose. Glucose could be linearly detected in the range from 0.1 to 2 mM with a LOD of 100 M. It is noted that, as compared with horseradish peroxidase, the BSA-Cu NCs exhibit higher activity near neutral pH, which is beneficial for biological applications. Zou et al. [68] developed a simple one-pot synthesis method for preparation of Cu NCs by employing dopamine (DA) as a reducing and capping reagent. Based on the peroxidase-mimicking catalytic features of the resulting Cu NCs, sensing of Fe3+ was realized because Fe3+ ions have specific interactions with the catechol groups on the surface of dopamine capped Cu NCs with a LOD of 4.2 mM. 7. Conclusion and future perspectives In summary, we presented an up-to-date review of the analytical applications of Cu NCs. Cu NCs are emerging as a very promising analytical platform for diverse sensing applications, especially for metal ions, biomoleculars (proteins, nucleic acids etc.), small molecules and pH. The main feature of such detection is the label-free characteristic of Cu NCs. More importantly, due to their ultrafine size, good biocompatibility and nontoxicity, luminescent Cu NCs have been increasingly investigated for biolabeling and bioimaging applications. This provides an opportunity to combine with other molecular imaging modalities for sensitively and accurately diagnostic research and biomedical applications. For example, through the conjugation of BSA to the tumor target peptide LHRH, Gao et al. [59] have demonstrated that [64Cu]CuNC@BSA-LHRH can be used as a tumor PET imaging agent for early, sensitive, and accurate diagnosis in a primary (orthotopic) lung cancer in vivo, due to their high radiolabeling stability, ultrasmall size, and rapid deposition 20 Page 20 of 30

and diffusion into tumor, as well as predominantly renal clearance. Although fluorescence-based molecular sensing with Cu NCs is a convenient way, it is of great interest to develop analytical applications of Cu NCs based on their catalytic properties. For example, chemiluminescent and electrochemical sensing with Cu NCs are relatively new [61,64] and may open a new field for the development of novel analytical methods and expand the utilization of Cu NCs. Additionally, although Cu NCs have demonstrated enormous potential in various analytical applications, they have some shortingcomings, for instance, the quantum yield (QY) of the reported Cu NCs is still usually low and much lower than that of semiconductor QDs. Thus, the facile synthesis of high quality Cu NCs via simple routes is still a challenge. In addition, Cu NCs promise a high tendency to undergo oxidation particularly at the subnanometer size regime. This makes their synthesis and purification much more challenging. We can foresee that the future of Cu NCs is highly related to the synthesis of high quality Cu NCs and diverse applications. Acknowledgments The financial support of the research by the Natural Science Foundation of China (No.21277111) and the Fundamental Research Funds for the Central Universities (XDJK2013D001) are gratefully acknowledged. References [1] H.M. Lee, M. Ge, B.R. Sahu, P. Tarakeshwar, K.S. Kim, Geometrical and electronic structures of gold, silver, and gold−silver binary clusters: origins of ductility of gold and gold−silver alloy formation, J. Phys. Chem. B 107 (2003) 999410005. [2] J. Zheng, P.R. Nicovich, R.M. Dickson, Highly fluorescent noble-metal quantum dots, Annu. Rev. Phys. Chem. 58 (2007) 409431. [3] D.I. Gittins, D. Bethell, D.J. Schiffrin, R.J. Nichols, A nanometre-scale electronic switch consisting of a metal cluster and redox-addressable groups, Nature 408 (2000) 6769. [4] J.D. Aiken III, R.G. Finke, A review of modern transition-metal nanoclusters: their synthesis, characterization, and applications in catalysis, J. Mol. Catal. A 145 (1999) 144. [5] L.B. Zhang, E.K. Wang, Metal nanoclusters: new fluorescent probes for sensors and bioimaging, Nano Today 9 (2014) 132157. [6] X. Yuan, Z. Luo, Y. Yu, Q. Yao, J. Xie, Luminescent noble metal nanoclusters as an emerging optical probe for sensor development, Chem.Asian J. 8 (2013) 858871. 21 Page 21 of 30

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for targeting orthotopic lung tumors using accurate positron emission tomography imaging, ACS Nano 9 (2015) 4976–4986. [60] X. Zhu, H. Shi, Y. Shen, B. Zhang, J. Zhao, G. Li, A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters, Nano Res. 8 (2015) 27142720. [61] H. Chen, L. Lin, H. Li, J. Li, J.M. Lin, Aggregation-induced structure transition of protein-stabilized zinc/copper nanoclusters for amplified chemiluminescence, ACS Nano 9 (2015) 21732183. [62] S. Xu, F. Chen, M. Deng, Y. Sui, Luminol chemiluminescence enhanced by copper nanoclusters and its analytical application, RSC Adv. 4 (2014) 1566415670. [63] S. Xu, M. Deng, Y. Sui, Y. Zhang, F. Chen, Ultrasensitive determination of bisphenol A in water by inhibition of copper nanoclusters-enhanced chemiluminescence from the luminol–KMnO4 system, RSC Adv. 4 (2014) 4464444649. [64] Z. Wang, L. Si, J. Bao, Z. Dai, A reusable microRNA sensor based on the electrocatalytic property of heteroduplex-templated copper nanoclusters, Chem. Commun. 51 (2015) 6305 6307. [65] Y. Li, J. Sun, C. Bian, J. Tong, S. Xia, A micro electrochemical sensor with porous copper-clusters for total nitrogen determination in freshwaters, 2013 8th IEEE International Conference on Nano/Micro Engineered and Molecular Systems (2013) 7679. [66] X. Kang, Z. Mai, X. Zou, P. Cai, J. Mo, A sensitive nonenzymatic glucose sensor in alkaline media with a copper nanocluster/multiwall carbon nanotube-modified glassy carbon electrode, Anal. Biochem. 363 (2007) 143150. [67] L. Hu, Y. Yuan, L. Zhang, J. Zhao, S. Majeed, G. Xu, Copper nanoclusters as peroxidase mimetics and their applications to H2O2 and glucose detection, Anal. Chim. Acta 762 (2013) 8386. [68] H.Y. Zou, J. Lan, C.Z. Huang, Dopamine derived copper nanocrystals used as an efficient sensing, catalysis and antibacterial agent, RSC Adv. 5 (2015) 5583255838.

26 Page 26 of 30

Table Caption

Table 1. A summary of photophysical properties and analytical applications of copper nanoclusters

Figure Captions Fig. 1. Schematic illustration of detection strategy based on the formation of fluorescent Cu NCs (Y: SNP site). (Reprinted with permission from [48], 2012, American Chemical Society) Fig. 2. Schematic diagram of the possible CL mechanism of the NaHCO3-Zn/BSA CuNCs-H2O2 system. (Reprinted with permission from [61], 2013, American Chemical Society) Fig. 3. Schematic illustration of oxidation color reaction of TMB by H2O2 catalyzed by Cu NCs as peroxidase mimetics and colorimetric detection of glucose by coupling glucose oxidase (GOx). (Reprinted with permission from [67], 2013, Elsevier)

Page 27 of 30

Table 1. A summary of photophysical properties and analytical applications of copper nanoclusters Target

Cu NCs material

Maximum em

UV-Vis

/nm (ex) /nm

absorption/nm

QY/%

Linear range

LOD

Ref.

110 nM

1 nM

[13]

0.11000 μM

0.024 μM

[14]

/

10 nM

[15]

10 pM10 μM

4.7 pM

[16]

/

/

[17]

Applications of Cu NCs for fluorescent sensing Hg2+

544(262,287)

/

cysteine-Cu NCs

480(375)

375

ssDNA-Cu NCs

480(345)

345

BSA-Cu NCs

420(320)

/

BSA-Cu NCs

410(325)

325

dsDNA-Cu NCs

585(340)

/

5100 nM

5 nM

[18]

TBAN-CuNCs

408(312)

/

/

4.9 µM

[21]

GSH-CuNCs

606(420)

/

1160 nM

1 nM

[22]

BSA-CuNCs

420(340)

325

0.0234 μM

1 nM

[23]

Cu

2+

DPA-CuNCs

673(391)

/

0.956.35 ppm

0.3 ppm

[24]

Cu

2+

PAA-g-MEA-CuNCs

630(360)

/

5.7

/

/

[25]

3+

BPEI-CuNCs

430(360)

355

2.1

0.51000 μM

340 nM

[26]

3+

Fe

TA-CuNCs

430(360)

230, 268, 290

14

0.01 10 μM

10 nM

[27]

Cr(VI)

cysteine-Cu NCs

490(360)

360

5.6

0.260 µM

65 nM

[28]

ONPs-Cu NCs

620(468)

272, 308, 400,

/

/

3 µM

[29]

0.0160 μM

5 nM

[30]

0800 μM

100 nM

[31]

0.07-80 μM

9.6 nM

[32]

130 μM

0.1 μM

[33]

2+ 2+ 2+

Pb

2+

Pb

2+

Pb

2+

Pb

2+

Cu2+

Hg Hg Hg

Fe

Cr

3+

CTAB-Cu NCs

14.3

15

5.3

431, 468 CN I





PO43 

MBA-Cu NCs

420(338)

/

13

PEI-Cu NCs

430(270)

/

TA-Cu NCs

430(360)

230, 268, 290

14

PVP-Cu NCs

446(370)

265

12.64

2

cysteine-Cu NCs

460(382)

/

/

0.250 μM

42 nM

[34]

ATP

dsDNA-Cu NCs

596(340)

/

/

0.05500 μM

28 nM

[20]

GSH, Cys, Hcy

dsDNA-Cu NCs

580(340)

/

/

Cys: 2100 μM;

25 μM

[35]

120 nM

[36]

4570 nM

[37]

ClO S

GSH: 280 μM; Hcy: 5200 μM 0.8100 μM

TNP

BSA-Cu NCs

400(325)

/

/

Sudan I-IV

PEI-Cu NCs

480(355)

268, 355

3.8

H2S

PA-Cu NCs

580(326)

/

2.0

1100 μM

500 nM.

[38]

H2O2

PEI-Cu NCs

480(355)

268, 355

3.8

0.5–10 μM

0.4 μM

[39]

glucose

PEI-Cu NCs

480(355)

268, 355

3.8

10–100 μM

8 μM

[39]

H2O2

DPA-Cu NCs

418,640 (345)

/

16.6

0.05–2 mM

0.01 mM

[40]

H2O2

DHLA-Cu NCs

325 (a broad

/

1–10 μM

0.3 μM

[41]

1–50 μM

0.2 μM

[42]

0.2–50 μM

0.07 μM

[43]

/

/

[44]

I,II:

0.130

μM;

III,IV: 0.125 μM

590(342)

absorption band) H2O2

Papin-CuNCs

620(370)

/

14.3

kojic acid

BSA-CuNCs

407(330)

216, 270

/

guanosine

histidine-Cu NCs

456(350)

440

1.6

5’-triphosphate

Page 28 of 30

Table 1. continued Target

Cu NCs material

Maximum em

UV-Vis

/nm (ex) /nm

absorption/nm

QY/%

Linear range

LOD

Ref.

melamine

Poly T-Cu NCs

598(360)

/

/

0.1– 6 μM

95 nM

[45]

ALP

dsDNA-Cu NCs

565(335)

/

/

0.1–2.5 nM

0.1 nM

[46]

exonuclease III

dsDNA-Cu NCs

575(350)

/

/

0.05–2 U mL-1

0.02 U mL-1

[47]

microRNA

dsDNA-CuNCs

608(340)

/

/

0.001 10 nM

1 pM

[49]

microRNA

concatemeric

570(340)

/

/

10 400 pM

10 pM

[50]

dsDNA-Cu NCs pH

GSH-Cu NCs

606(420)

/

5.3

49

/

[22]

pH

BSA-Cu NCs

620(524)

/

4.1

612

/

[52]

pH

trypsin-Cu NCs

455(363)

350

1.1

2.0212.14

/

[53]

pH

BSA-Cu NCs

420(320)

/

/

214

/

[54]

cellular imaging

lysozyme-Cu NCs

450(360)

/

18

/

/

[55]

temperature

CLEDNN-Cu NCs

454(373)

/

7.3

/

[56]

cellular imaging

Trf-Cu NCs

670(508)

/

6.2

/

/

[57]

cellular imaging

GSH-Cu NCs

430(340)

/

6

/

DNA staining

DNA-Cu NCs

584(343)

380

/

/

430(350)

350

/

10 C55 C

[58] /

[60]

0.005–1 μM

0.3 nM

[61]

Applications of Cu NCs for chemiluminescent sensing H2O2

BSA-Zn/Cu NCs

H2O2

BSA-Cu NCs

/

325

/

0.1150 mM

0.03 mM

[62]

bisphenol A

BSA-Cu NCs

/

325

/

0.00110 μM

0.12 nM

[63]

Applications of Cu NCs for electrochemical sensing microRNA

DNA-RNA Cu NCs

/

/

/

25300 fM

8.2 fM

[64]

glucose

Cu NCs-CNTs

/

/

/

0.00073.5 mM

0.21 μM

[66]

Applications of Cu NCs for colorimetric sensing H2O2

BSA-Cu NCs

/

/

/

0.1150 mM

0.03 mM

[67]

glucose

BSA-Cu NCs

/

/

/

0.1–2 mM

100 μM

[67]

DA-Cu NCs

390(320)

/

9.6

5–300 μM

1.2 μM

[68]

3+

Fe

/: not indicated or mentioned.

Page 29 of 30

Fig. 1

Fig. 2

Fig. 3

Page 30 of 30