Effects of protecting groups on luminescent metal nanoclusters: spectroscopic signatures and applications

Abstract

Luminescent metal nanoclusters (NCs) have been established as next-generation fluorophores. Their biocompatible and non-toxic nature, along with excellent chemical- and photo-stability, enables them to find applications in multi-disciplinary areas. However, preparing NCs which are stable is always challenging, primarily owing to their small size and propensity to self-aggregate. In this review, we highlight a holistic approach as to how ligands and templates can monitor the stability of NCs, tune their spectroscopic signatures, and alter their applications. The role of small molecules of a large ligand in the preparation of NCs and their associated limitations are also discussed. We have summarized how these NCs can be utilized in sensing several metal ions, pH, viscosity and temperature of many systems which have biological relevance. Additionally, these luminescent metal NCs find usage in cell-imaging, discriminating between cancerous and non-cancerous cell lines and also targeting specific organelles within the cellular environment.

---

Subhajit Chakraborty

Subhajit Chakraborty completed his BSc with Chemistry majors from the Ramakrishna Mission Vivekananda Centenary, Kolkata, India, and received his MSc degree from the Presidency University, Kolkata. He has joined PhD program in the Department of Chemistry, IISER Bhopal, under the supervision of Professor Saptarshi Mukherjee. He is primarily working on luminescent metal nanoclusters, and their applications in biological systems using spectroscopic and microscopic techniques. He loves to paint and write in his leisure time.

Saptarshi Mukherjee

Professor Saptarshi Mukherjee received his BSc and MSc degrees from Jadavpur University, Kolkata, India. He carried out his PhD under the supervision of Professor Kankan Bhattacharyya at IACS, Kolkata, and post-doctoral research with Professor H. Peter Lu at the Bowling Green State University, Ohio. He joined the Department of Chemistry, IISER Bhopal, in 2008. His research interests include luminescent metal nanoclusters, protein unfolding and refolding dynamics, and surface chemistry using spectroscopic and microscopic approaches. He is the recipient of INSA Young Scientists Award, CRSI Bronze Medal, CRSI Young Scientist Award and is a Senior Editor of Chemical Physics Impact. He is also a founding member of the Indian National Young Academy of Science.

---

Introduction

Recently, ultra-small-sized metal nanoclusters (NCs) have drawn considerable research interest owing to their unique size-dependent characteristics consequent to the quantum confinement effect.^1–14 These metal NCs are characterized by easy synthetic protocols, high photostability, water solubility, catalytic properties, biocompatibility, non-toxicity, etc., which enable them as next-generation fluorophores.^15–30 Generally, metal NCs are very robust to photon irradiation; they are much more photostable as compared to commercial organic dyes.^13,15 This thereby enables them to be used in single-molecule spectroscopic investigations where photobleaching is one of the challenges that need to be overcome. Additionally, these metal NCs are generally non-toxic and biocompatible.^13,19,29 This feature of metal NCs differentiates them from analogous quantum dots (QDs) which are generally far more toxic. Due to these interesting features, metal NCs can be utilized for a plethora of applications in interdisciplinary research fields and thereby can act as a replacement for organic dyes and quantum dots (QDs). However, despite these advantages, there are some bottlenecks related to metal NCs which require further exploration and refinements. One of the major challenges is related to the stability of these metal NCs after their formation. Lack of proper surface protection can lead to the oxidation of metal atoms present in the core of NCs which results in their degradation. Often, larger nanoparticles are generated along with NCs which are mostly non-fluorescent. Consequently, an inherent heterogeneity appears within the system. For this reason, the selection of capping ligands and the optimization of synthetic protocols must always be properly addressed. Another shortcoming of these metal NCs is their low quantum yields (QY) as compared to those of QDs and conventional fluorophores. Although investigations are going on to circumvent this aspect, it requires more exploration so that these metal NCs can be used in applications that require greater photoluminescence intensities.

The optical properties of larger metal nanoparticles (NPs) are strongly dependent on the free electron density and their sizes, which can be explained by the Mie theory.^31–33 The Mie theory can still provide an understanding for the particles having a size comparable to the electron mean free path.^31,34 However, interestingly, when the particle size approaches the Fermi wavelength of the conduction electron (i.e., formation of NCs), the optical properties become drastically different from the larger metal NPs as the continuous electronic band structure is converted into quantized discrete energy levels.^35–39 Consequently, these NCs become luminescent and act like molecular species. According to the Drude model,¹ the free electrons of the metal atoms (valence s-orbital for noble metals) become delocalized over the cluster core. The total Hamiltonian of the metal NCs may be expressed as a combination of the noninteracting Hamiltonians of the free electrons.¹ This concept is based on the “one-particle approximation” and is related to the particle-in-a-box model which explains the behaviour of free electrons in metals.¹ According to Hooke's law, a linear restoring force when applied on free electrons makes the quantized energy levels behave as three-dimensional harmonic oscillators.^40–42 Using the concept of free electrons based on the Drude theory, the Jellium model has been introduced to estimate the cluster–core composition which actually governs the luminescence properties of the said metal NC.¹⁶ A mathematical expression of the most accepted Jellium model, which connects the Fermi energy of the particular metal (E_Fermi), the corresponding emission energy (E_em) and the number of atoms in a given cluster–core (N), is as follows:¹⁶

E_em= E_Fermi/N^0.3 (1)

The most unique feature of metal NCs is their size-dependent luminescence properties. By tuning the size, one can regulate the photophysical characteristics of metal NCs.⁴³ However, various other factors like capping ligand and the environment around the cluster core have an enormous influence on the photophysical properties of metal NCs (will be discussed later).⁴³ Thus, the optical properties of metal NCs do not follow a linear relationship with the size of the NC alone.^43,44 Due to the presence of free electrons and their collective oscillations, NPs display characteristic surface plasmon resonance (SPR) bands in the UV-Vis absorption profile. This feature differentiates them from metal NCs. The formation of discrete energy levels due to the reduction in size during the formation of metal NCs facilitates the one-electron transition in between quantized energy states. Consequently, a stepwise optical absorption results in a continuous absorption signature. Interestingly, the concept of enhancement of luminescence intensity through aggregation has been explained in recent times. In 2012, Xie and co-workers reported the formation of bright emitting AuNCs and their luminescence properties have been ascribed to the concept of aggregation-induced emission (AIE).⁴⁵ In contrast to larger sized non-luminating NPs, AIE provides the attachment of the neighbouring protecting ligands of the individual cluster–cores with one another.^7,46 Thus, the AIE phenomenon is associated with the large Stokes shift and long excited-state lifetime decay components.⁴⁶

There are various ways by which NCs can be synthesized, and such protocols are well-documented.^47,48 Generally, two such standard approaches are followed: (i) top-down approach and (ii) bottom-up approach (Scheme 1). The top-down approach involves some dry and wet grinding/etching processes. Additionally, the yields of the final products are generally low which thereby limits this approach toward the formation of stable and cost-effective NCs. Hence, the favoured approach is the bottom-up approach, which is a combination of reduction of metal ions to their zero-valent oxidation states followed by the aggregation of the reduced metal atoms and encapsulation by ligands which leads to the formation of stable metal NCs.¹² In general, this process involves two steps: (i) bio-mineralization;^12,49 reduction of metal ions in the presence of reducing agent(s) (sometimes the ligand itself can serve this purpose) which produces polydispersed cluster–cores⁵⁰ and (ii) subsequent nucleation; formation of molecularly pure cluster–cores. Very recently, Xie's and Ying's groups well described the 2e⁻ reduction process in a stepwise manner for the growth of cluster–cores of AuNCs containing Au⁰.^51–53 Sometimes the pH of the medium activates the ligand molecule or particularly, the functional group of the ligand molecule by deprotonation to exhibit the reducing ability.^13,15

Scheme 1 Schematic representation of the two different ways by which NCs can be prepared: (A) top-down approach and (B) bottom-up approach.

To avoid the aggregation of tiny clusters once formed and the subsequent formation of bigger-sized NPs, the ligands play a pivotal role by protecting the zero-valent cluster cores.^54,55 Not only the protection and surface stabilization of the cluster–core, but also the photophysical and morphological properties of NCs are also highly influenced by the capping agents.^56,57 To date, a large variety of ligands have been used for the facile preparation of noble metal (Au, Ag and Cu) NCs, such as protein, peptides, polymers, DNA, small molecules, etc. (Scheme 2).⁸

Scheme 2 Representation of different ligands used for the preparation of NCs.

In 1978, Bartlett and co-workers prepared 0.8 nm sized gold nanoclusters (AuNCs) using an amino-substituted triarylphosphine ligand (Ar₃P)₇Au₁₁X₃).⁵⁸ They observed that when the substituent, “X” was iodine, the clusters so formed were unstable and had a tendency to undergo oxidative decomposition.⁵⁸ However, Hutchison and co-workers demonstrated that stable AuNCs having a similar core composition (i.e., Au₁₁) can be prepared when alkanethiols are used as a capping agent instead of triarylphosphine ligands.⁵⁹ In a seminal work, Yang and Chen demonstrated that the increment of particle band gap energy (from 1.4 to 1.8 eV) can be achieved for gold particles by altering the capping ligand from triphenylphosphines to alkanethiolates.⁶⁰ Zhu and co-workers very nicely demonstrated that selenolate capped Au₂₅(SePh)₁₈⁻ NCs exhibited totally different electronic distribution and optical properties in comparison to its thiolate counterpart (Fig. 1), Au₂₅(SCH₂CH₂Ph)₁₈⁻, despite having a similar icosahedral Au₁₃ core and semi-ring motifs (Au₂(SeR)₃ or Au₂(SR)₃) (Fig. 2).⁶¹ Thus, the rational use of ligands can alter the HOMO–LUMO energy gap of the molecule.

Fig. 1 (A) Theoretical optical curves of Au₂₅(SePh)₁₈ and Au₂₅(SC₂H₄Ph)₁₈ nanoclusters as marked in the figure. Comparison of the component of KS orbitals of Au₂₅(SePh)₁₈ (B) and Au₂₅(SC₂H₄Ph)₁₈ (C) nanoclusters. Reproduced with permission from ref. 61. Copyright 2014, The Royal Society of Chemistry.

Fig. 2 Crystal structures of Au₂₅(SePh)₁₈⁻ (A) and Au₂₅(SC₂H₄Ph)₁₈⁻ (B) nanoclusters. (C) The Au₁₃ core of Au₂₅(SePh)₁₈⁻. (D) and (E) The distortions of Au₂Se₃ and Au₂S₃ motifs, respectively, in the σ_h plane along x–y (color labels: yellow = Au, violet = Se, orange = S, for clarity C and H atoms are omitted). Reproduced with permission from ref. 61. Copyright 2014, The Royal Society of Chemistry.

Also, the facile binding affinity (between the electron-donating atom of the ligand and metal ions/atoms) leads to the selectivity of ligands toward the formation of NCs. For example, the soft–soft interaction between the S atom and Au atom leads to the formation of highly stable AuNCs.^62,63 Using Raman spectroscopy, Duguid et al. monitored the interaction of DNA and a series of divalent metal atoms and demonstrated that the nitrogen centers of the nucleobases are more favourable toward bond formation with metal ions.⁶⁴ In two separate studies, Ono et al. and Ritchie et al. had opined that the cytosine-rich (C-rich) sequence of DNA promotes the easy formation of AgNCs as cytosine has a high affinity to bind with Ag⁺ ions.^65,66

In a recent study, Chevrier et al. investigated how the template influences the structure and properties of Au₁₈(SR)₁₄ NCs (where, –SR = cyclohexanethiolate ligand and glutathione ligand) using synchrotron X-ray spectroscopy and quantum mechanics/molecular mechanics (QM/MM) simulations (Fig. 3).⁵⁷ This report clearly demonstrated that encapsulation by glutathione can provide more stability through compact surface construction with suitable rigidity (Fig. 4). Also, the enhanced photoluminescence and biocompatibility of these AuNCs have been explained based on the above-mentioned concept.⁵⁷

Fig. 3 The plots represent the Au L₃-edge WT-EXAFS for (A) Au₁₈(SC₆H₁₁)₁₄ in toluene and (B) Au₁₈(SG)₁₄ in water. Reproduced with permission from ref. 57. Copyright 2018, American Chemical Society.

Fig. 4 (A) Simulated structures of Au₁₈(SC₆H₁₁)₁₄ in the gas phase and toluene phase, respectively, obtained from QM and QM/MM simulations. (B) Simulated structures of Au₁₈(SG)₁₄ in the gas phase and water phase, respectively obtained from QM and QM/MM simulations. Color labels: pink atoms = Au, yellow sticks = S, and cyan sticks = C. In (A), the toluene molecules of the first solvation shell are represented in black (carbon) and white (hydrogen). In (B), the water molecules of the first solvation shell are shown in red (oxygen) and white (hydrogen). Reproduced with permission from ref. 57. Copyright 2018, American Chemical Society.

Noble metal NCs have been widely used in sensing applications due to their selective and sensitive detection ability towards various chemical entities.^63,67–74 This property of these next-generation fluorophores enables them to be used for both in vitro and in vivo sensing. It is always challenging to sense chemical entities inside complex biological systems as they have direct relevance to biomedical investigations.⁶⁷ Discriminating cancerous and non-cancerous cells is extremely important for the early detection of diseases. It has been reported that AuNCs, templated by the globular protein (human serum albumin, HSA; bovine serum albumin, BSA) can be effectively utilized to differentiate between cancerous and non-cancerous cell lines.^62,63 The uptake of these AuNCs is much more in the case of cancerous cells as compared to the non-cancerous ones. Additionally, targeting specific organelles within a cell is also important from the perspective of drug delivery and subsequent release. The associated dynamics inside a cell are primarily controlled by the microenvironment viscosity, polarity, pH, etc. To have an in-depth understanding of the dynamics inside a cell and probe the microenvironment in and around an organelle, luminescent metal NCs are widely used.^75–78 The fact that these NCs are photostable, non-toxic and biocompatible in nature enables them to be efficiently used in single-molecule spectroscopic studies and for bioimaging investigations.^{62,63,79–81} For most of the cases, specific targeting of organelles within a cell can be realized by rational use of ligands.⁶⁷ It is pertinent to be mentioned that the translational diffusion of NCs is not similar in every cell due to the different microenvironment and system-specific localization of NCs inside the cell lines.^62,81 Bhattacharyya and co-workers studied the translational relaxation of AuNCs using fluorescence correlation spectroscopy (FCS) (Fig. 5).⁶² They demonstrated that the AuNCs follow 2D diffusion in the non-cancerous MCF10A cell lines (i.e., diffusion through the cell membrane only), whereas the same set of NCs experienced a combination of 2D and 3D diffusion in the cancerous MCF7 cell lines (i.e., localization both in the membrane and the cytoplasm, Fig. 5).⁶²

Fig. 5 Confocal images of breast cells stained with BSA-templated AuNCs. The degree of staining is much more for cancerous cells as compared to non-cancerous cells. (A) Normal breast cell (MCF10A) and (B) breast cancer cell (MCF7). (C) Normalized FCS traces of these AuNCs in bulk water (black), inside the cell membrane of MCF10A (blue), inside the cytoplasm of MCF7 (green), and in the cell membrane of MCF7 (red). Reproduced with permission from ref. 62. Copyright 2014, American Chemical Society.

Additionally, NCs are the suitable choice for the sensitive detection of pH,^82–84 various biomolecules such as proteins,⁸⁵ nucleic acids,^86,87 small biomolecules⁶⁷ and even viruses.^74,88 Triulzi et al. were the first to report the detection of human IgG using PAMAM coated AuNCs which were used as a fluorescence probe.⁸⁹ In another study, Chen et al. observed the rapid detection ability of GSH templated AuNCs for the detection of glutathione S-transferase (GST)-tagged proteins.⁹⁰ Using DNA-templated AgNCs, Martinez and co-workers reported the detection of a specific protein, thrombin, with an impressive detecting limit (1 nM).⁹¹ Digestion of proteins is mainly controlled by the protease enzyme which basically breaks the peptide bonds of the proteins. Wang et al. developed AuNCs using BSA as a capping ligand and demonstrated the sensitive nanoscale detection of protease (detection limit of 1 ng mL⁻¹) by turn-off emission signal phenomena.⁹² In another work, Zhu and co-workers prepared DNA-protected AgNCs which can act as a sentient towards the determination of adenosine deaminase (ADA).⁹³

Detecting toxins within biological systems and that too at low concentrations will certainly be useful. Nair et al. strategically prepared urease encapsulated NIR emitting AuNCs and demonstrated that these AuNCs can be utilized to selectively detect urea in human blood and cow milk samples.⁹⁴ Xie et al. developed GSH stabilized AgNCs for the sensitive and specific detection of cysteine (Cys), a sulfur-containing amino acid with the detection limit of <3 nM.⁹⁵ It is already established that a certain level of reactive oxygen species (ROS) is important for the normal functioning of a cell. However, the generation of excessive ROS inside the cell can easily affect this severely which may, in turn, lead to permanent damage of the cell. We have recently developed a potential antimicrobial agent (tyrosine templated silver nanomaterials, AgNMs) which can show the prompt killing of cell lines by creating a high level of ROS inside the cells.⁹⁶ On the other hand, detection and quantification of ROS is equally important for therapeutic applications. Chen et al. developed a dual emissive AuNC decorated silica nanosensor for the efficient detection of highly ROS (hROS) inside the live HeLa cell lines (Fig. 6).⁹⁷

Fig. 6 Detection of hROS using the crown nanoparticle assembly. The system consists of a core dye encapsulated silica particle and multiple satellite glutathione-templated AuNCs. Reproduced with permission from ref. 97. Copyright 2013, American Chemical Society.

Additionally, NCs can be utilized for the efficient detection of various inorganic cations and anions. In a recent work, Nain et al. synthesized AuNCs, AgNCs and CuNCs using a mixture of BSA and thiosalicylic acid (TSA) as the encapsulating ligands.⁹⁸ Interestingly, they have shown that each of these NCs can selectively detect a metal ion(s); AuNCs, AgNCs, and CuNCs can separately detect Hg²⁺, As³⁺ and Cr⁶⁺ ions, respectively.⁹⁸ We have demonstrated that HSA templated AuNCs can show ultra-sensitive and selective detection of Hg²⁺ ions in vitro as well as in vivo environments.⁶³ In a previous study from our group, Ghosh et al. demonstrated that HSA-templated Ag₉:HSA NCs can sense Co²⁺ ions by a turn-off luminescence mechanism.⁹⁹ Interestingly, these quenched NCs can again detect Zn²⁺ ions in solution by restoring the luminescence intensity (turn-on luminescence mechanism).⁹⁹ Chang and co-workers¹⁰⁰ developed DNA-templated AgNCs for the selective detection of Cu²⁺ ions in Montana soil (SRM 2710) and pond water. GSH-capped CuNCs displayed the selective sensing ability towards Fe³⁺ ions with a low limit of detection (LOD) of ∼25 nM.¹⁰¹ Cytidine-stabilized AuNCs have the ability for the selective detection of Ag⁺ ions in solution by turn-on fluorescence,¹⁰² whereas Pb²⁺ ions can also be detected by GSH-templated AgNCs with a LOD of ∼0.2 nM.¹⁰³ Additionally, metal NCs can serve as efficient biomarkers.^19,63,81 The easy encapsulation of NCs inside the cell lines and their bio-imaging abilities have been well-documented.^19,63,81,93 Sun et al. fabricated NIR luminescent AuNCs using GSH as a capping and reducing agent and demonstrated that further conjugation of folic acid rendered these NCs as efficient biomarkers through encapsulation inside HeLa cell lines.¹⁰⁴ A ligand comprising 18 peptides has been used to prepare AuNCs characterized by bright emission at the NIR region (1100–1350 nm). These NCs were subsequently utilized for in vivo brain vessel imaging and tumour metastasis.¹⁰⁵ Recently, Xie and co-workers developed cyclodextrin (CD) templated AuNCs exhibiting NIR-II emission (λ_em = 1050 nm) which have thereby been used as a NIR-II imaging probe for in vivo investiogations.¹⁰⁶

In our research work, we have used various kinds of ligands, such as proteins (human serum albumin, HSA), tripeptides (reduced glutathione, GSH), small molecules (tyrosine, Tyr), etc., to prepare several metal NCs.^{63,72,80,96,99,107} We have prepared AgNCs having two different compositions (Ag₉:HSA and Ag₁₄:HSA) using HSA as a template and for the first time demonstrated that NCs can be toggled/interconverted using simple redox chemistry.^107,108 Using the same template HSA, we have also reported that CuNCs can exhibit thermal sensitivity and act as a nanothermometer. Additionally, these CuNCs serve as a FRET pair with the standard dye Coumarin-153, characterized by an efficiency of energy transfer of ∼85%.¹⁰⁹ We have also prepared CuNCs using GSH as a capping agent.¹⁰⁴ These CuNCs act as a biomarker for cancerous cells and also selectively detected Fe³⁺ ions in solution and the protein haemoglobin, which in turn can lead to the early detection of cyanosis.¹⁰⁴ In our investigations on HSA-capped AgNCs and CuNCs, no external reducing agent was used. This was achieved by keeping the pH of the medium greater than the pK_a of tyrosine.¹⁵ In these studies, we had proposed that the 18 tyrosine amino acid residues inherently present within the protein matrix provide the necessary reducing environment (at a pH ∼ 11, i.e., greater than 10.46, which is the pK_a of tyrosine^15,107) and result in the formation of stable NCs. To substantiate our proposal, we developed a unique strategy of using bare L-tyrosine as a capping agent outside the protein environment to prepare stable NCs.¹¹⁰ These investigations not only highlight the role of a small molecule of a large ligand in preparing stable NCs, but also clearly demonstrate the advantages of having larger templates as compared to smaller ones as the former reduces the extent of heterogeneity within the system.¹¹⁰ This feature article highlights the various fundamental aspects and related applications of luminescent metal NCs. The field of cluster chemistry is developing extremely rapidly and encompasses various other interdisciplinary research objectives. Since the properties of luminescent metal NCs greatly depend on the choice of ligands used, we have in detail delineated the effect of ligands on the spectroscopic signatures of these NCs. Besides the fundamental physics/chemistry involved with their preparation, metal NCs have been established as next-generation fluorophores owing to their tunable size-dependent luminescence properties. Herein, we have discussed how these NCs can be used in bio-medics, sensing metal ions with unprecedented limits of detection, sensing the pH and viscosity of cellular environments, and serving as bio-markers in differentiating both cancerous and non-cancerous cell lines.

Effects of protecting ligands

As mentioned earlier, for the preparation of stable metal NCs, generally, the “bottom-up” approach is preferred. In this process, reduction of metal ions to zero-valent metal atoms followed by cluster–core formation and encapsulation by ligands are the key factors that lead to the formation of NCs. Generally, the cluster–core, comprising metal atoms in their zero-valent oxidation states, has a strong propensity to self-aggregate owing to a very high surface energy. This in turn leads to the formation of larger-sized nanoparticles. However, to restrict further aggregation (i.e., to prevent the formation of nanoparticles from NCs), ligands play a vital role and offer the necessary surface stability.^54,55 Not only the formation, but also the long-time stability, photophysical properties, etc. can be regulated by capping agents.^63,109,110 In general, the compactness around the cluster–core can provide extra surface stability to NCs.^63,109 Recently, small molecules (like thiolates, amino acids, etc.) have been widely used for the preparation of stable NCs.^101,110 However, macromolecular ligands generally provide extra surface rigidity that is required to ensure the formation of a NC, which is more robust and exhibits greater photo-, thermal- and chemical stability.^63,109

We have prepared blue light-emitting CuNCs using the globular protein HSA as a capping as well as reducing agent.¹⁰⁹ HSA is the most abundant unglycosylated monomeric circulatory protein found in the human blood plasma and is produced in the liver.^111,112 The average molecular mass of HSA is 66.4 kDa and this protein consists of 585 amino acid residues in a single poly-peptide chain.¹¹³ Ghosh et al. proposed that the Tyr amino acid residues which are present in the HSA scaffolds are actually responsible for the formation of NCs.¹⁰⁹ Bao and co-workers also reported that the Tyr act at the final step in reducing Au⁺ ions to Au atoms during the preparation of AuNCs using BSA as a protective ligand.¹¹⁴ In these synthetic protocols, the pH of the reaction medium is maintained at ∼11 which renders the Tyr to behave as a reducing agent as the pK_a of Tyr is 10.46.^15,107 These CuNCs exhibited a strong luminescence intensity centered at ∼414 nm upon excitation at 330 nm. However, this spectroscopic signature was almost completely lost when these CuNCs were treated with a strong oxidant, H₂O₂. Here, H₂O₂ oxidizes the Cu⁰ from the cluster–core to Cu⁺ or Cu²⁺ which eventually results in the diminution of luminescence intensity.¹⁰⁹ The time-resolved decay transients of these CuNCs exhibited a bi-exponential decay [1.10 ns (30%) and 3.40 ns (70%)]. This bi-exponential nature of the decay transient has been attributed to the electronic transitions involved between the filled d orbitals and the vacant sp conduction band. The longer component of 3.40 ns has been ascribed to the transition between Cu and the ligand (here HSA), while Cu–Cu interactions account for the shorter component of 1.10 ns.¹⁰⁹ Through time-dependent emission profiles, it has been demonstrated that the formation of stable CuNCs is a slow process with the entrapped Cu²⁺ ions getting stabilized through electrostatic interactions involving several functional groups and the bulky structure of the protein. Besides showing thermal sensitivity and reversibility, these CuNCs demonstrated temperature-dependent FRET. The CuNCs served as the donor while the acceptor was Coumarin153, non-covalently attached to the protein matrix (Scheme 3). The protein, HSA, is a heart-shaped molecule having three domains, I, II and III, each having two sub-domains, A and B.¹¹³ Molecular docking study suggested that the probe, Coumarin153, gets attached at the subdomain IB of HSA (probable location being near the Cys-34 amino acid residue of the protein) and attains stabilization through hydrophobic and electrostatic interactions.¹⁰⁹ The distance between the donor (CuNCs) and the acceptor (Coumarin153) estimated from molecular docking was ∼20 Å, while that estimated from FRET-based assay was ∼ 19 Å (Fig. 7).¹⁰⁹

Scheme 3 (A) Schematic representation displaying the phenomenon of FRET (donor: CuNCs; acceptor: Coumarin153) and nanothermometric behaviour exhibited by HSA-templated CuNCs. Reproduced with permission from ref. 109. Copyright 2015, American Chemical Society.

Fig. 7 (A) Molecular docking results representing the minimum-energy conformations of HSA upon binding with Coumarin153 (non-covalently attached to subdomain IB of HSA) and the plausible location of the binding of the fluorophore, Coumarin153. (B) Zoomed-in view displaying the several amino acid residues which are present in the immediate vicinity of Coumarin153 and the estimated distance between the acceptor (Coumarin153) and Cys-34 (20.2 Å). Reproduced with permission from ref. 109. Copyright 2015, American Chemical Society.

In a different work, Anand et al. prepared two AgNCs having different compositions, but using the common capping agent/template, i.e., HSA.¹⁰⁷ For the preparation of both these AgNCs, the pH of the medium was maintained greater than 10.46, i.e., the pK_a of Tyr,¹⁰⁷ and the temperature was maintained at 37 °C. However, the time taken for synthesis vastly differed; Ag₉:HSA was prepared in 10 hrs and no external reducing agent was used, while the other NC, Ag₁₄:HSA, was prepared within 2–3 minutes as in this case, and a strong reductant, NaBH₄, was used (Scheme 4). Since the composition differed, so did their spectroscopic signatures; while Ag₉:HSA (QY ∼ 16%) emitted at 460 nm, Ag₁₄:HSA (QY ∼ 11%) exhibited a strong luminescence at 620 nm.¹⁰⁷ The most interesting aspect of this investigation was the interconvertibility among these NCs (Fig. 8). Using simple redox chemistry, it has been demonstrated that these NCs can be toggled retaining almost all their photophysical properties (Scheme 4). These results highlight the role played by a bulky ligand. HSA provides the necessary protective environment as the cluster–cores do not get destroyed when a strong reductant (NaBH₄) and an oxidant (H₂O₂) were used to carry out the toggling between the two NCs.¹⁰⁷

Scheme 4 Schematic representation showing the formation and interconversion of the Ag₉:HSA and Ag₁₄:HSA NCs using redox chemistry. The two NCs were prepared independently using two different synthetic protocols. Reproduced with permission from ref. 107. Copyright 2012, American Chemical Society.

Fig. 8 Normalized fluorescence excitation spectra of the two HSA-templated AgNCs. The similar nature of these excitation spectra corroborates the fact that even after inter-conversion (from Ag₉:HSA to Ag₁₄:HSA and vice-versa), both the NCs retain their spectroscopic signatures signifying that upon inter-conversion, there are no major structural alterations. Reproduced with permission from ref. 107. Copyright 2012, American Chemical Society.

Besides surface stabilization, ligands can also influence the photophysical properties of NCs. Zhang and co-workers prepared carboxylate-protected AgNCs which showed excitation-dependent emission (Fig. 9A).¹¹⁵ Similar observation has been reported for DNA-coated AgNCs by Shao and co-workers (Fig. 9B).¹¹⁶ Previously, this phenomenon was ascribed to the formation of heterogeneity (i.e., the presence of NCs with different sizes) in the system.^117,118 However, Zhang and co-workers first explained this observation based on the surface plasmon and emitter coupling.¹¹⁵ In these AgNCs, the luminescence was controlled by the coupling between ligand-to-metal−metal charge transfer (LMMCT), i.e., from the complex Ag(I)–carboxylate to the core Ag atoms (Fig. 9C). Coupling between the surface plasmon and emitter can generate a new energy state which can be mathematically expressed as follows:¹¹⁶

Fig. 9 (A) Excitation-dependent emission exhibited by carboxylate-templated AgNCs using PMAA as a scaffold, owing to a strong coupling between the surface plasmon and the emitter. (B) Excitation-dependent emission profiles of the DNA-templated AgNCs. The samples were excited from 440 nm to 650 nm, with an interval of 10 or 20 nm. (C) Structural illustrations of luminescent Ag–carboxylate with Ag⁺–carboxylate complex shell. (D) Emission spectra of Tyr-templated AgNCs as a function of excitation wavelengths as marked in the figure. (A) and (C) Reproduced with permission from ref. 115. Copyright 2014, American Chemical Society. (B) Reproduced with permission from ref. 116. Copyright 2015, American Chemical Society. (D) Reproduced with permission from ref. 96. Copyright 2021, American Chemical Society.

Here, E_PL(λ) represents the uncoupled surface plasmon energy and λ is the excitation wavelength, with E_PL and λ expressed in eV and nm, respectively. E₀ denotes the transition energy of the ligand to metal charge transfer (LMCT) and Δ is the energy related to the interactions between the photons and electrons.¹¹⁶ This coupling process can also influence the QY and excited-state lifetime of NCs. On the other hand, when we prepared AgNCs using L-Tyr as a template, no such excitation-dependent emission profiles were observed (Fig. 9D).⁹⁶ In this case, the emission wavelength did not alter with a change in excitation wavelength, and only a change in emission intensity was obtained, as expected. Thus, it can be rationalized that the template can play a pivotal role in facilitating the plasmon-emitter coupling phenomenon.

The surface charge of any particular ligand also has a profound influence on the properties of metal NCs.¹¹⁹ Shang and co-workers strategically engineered the surface charged groups of BSA molecules for the preparation of different AuNCs.¹¹⁹ They designed two different types of BSA molecules from normal BSA (nBSA): one having more negative net charges (aBSA) and another one having more positive net charges (cBSA). Zeta-potential study confirmed the successful transformation of these two types of BSA molecules.¹¹⁹ Interestingly, the spectroscopic signatures of these (a/n/c)-BSA-templated AuNCs were very different (Fig. 10A). The following order was observed both in terms of luminescence intensities (all being red-emitting) and excited-state lifetimes: (cBSA capped AuNCs) > (nBSA capped AuNCs) > (aBSA capped AuNCs) (Fig. 10B).

Fig. 10 (A) Photographic images of (c/n/a)-BSA-templated AuNCs. The left panel represents the photographs when viewed under ordinary daylight while the right panel represents the same when viewed under a 365 nm UV lamp. (B) Luminescence profiles of different BSA-templated AuNCs as marked in the figure. (C) Cell viability assay of HeLa cells when incubated with (0 mg mL⁻¹, 250 mg mL⁻¹, 500 mg mL⁻¹ and 750 mg mL⁻¹) (c/n/a)-BSA-templated AuNCs for 4 hours. (D) Uptake efficiency of (c/n/a)-BSA-templated AuNCs after incubation with HeLa for 4 hours. (E) Pictorial representation of the cellular uptake of BSA-templated AuNCs with low and high charge densities. Reproduced with permission from ref. 119. Copyright 2020, The Royal Society of Chemistry.

Generally, the formation of AuNCs is initiated by the complex formation between Au(I) and the ligand (in this case, ligands are cBSA and aBSA molecules). It is expected that this complex formation could be more facile for the positively charged amino groups as the salt of Au is in AuCl₄⁻ form in the solution phase. Also, the emission maximum for cBSA capped AuNCs was observed to be more blue-shifted as compared to the aBSA capped AuNCs (Fig. 10B). Based on the Jellium model, one can expect that the number of metal atoms comprising one NC will be more in the case of aBSA capped AuNCs as compared to the cBSA capped AuNCs. In fact, the TEM micrographs reported by Shang and co-workers also support the same conjecture.¹¹⁹ Additionally, these two AuNCs demonstrated different cellular uptake and viabilities (Fig. 10C–E).¹¹⁹ Inductively coupled plasma-optical emission spectrometry (ICP-OES) suggested that the encapsulation of these AuNCs inside cell lines was around 2.5-fold higher for the aBSA capped AuNCs compared to the other two AuNCs (Fig. 10D). In this regard, the non-specific interaction of negatively charged (ζ = −31.6 mV) AuNCs with positive sites of the cell membrane favoured the endocytosis of AuNCs (Fig. 10E).¹¹⁹

In another work, Song et al. demonstrated that the capping ligand can also regulate the electrochemical properties of NCs despite their similar core crystal structures.⁶¹ Crystal structure determination revealed that the icosahedral Au₁₃ core and semi-ring Au₂(XR)₃ motifs (where, X = Se and S) are common for both AuNCs. They have determined the electrochemical properties of selenolate-templated Au₂₅ clusters using square wave voltammogram (SWV) experiments and outlined the differences with the previously reported thiolate-capped Au₂₅ clusters.⁶¹ While the Au₂₅(SePh)₁₈ NCs displayed four reduction peaks (R1, R2, R3 and R4), the Au₂₅(SCH₂CH₂Ph)₁₈ NCs exhibited only two reduction peaks (R1 and R2) (Fig. 11).⁶¹ Also, thiolate protected AuNCs were characterized by a greater electrochemical HOMO–LUMO energy gap (∼1.62 V) as compared to selenolate protected AuNCs (∼1.24 V). Furthermore, the gap between two successive oxidation states (O1 and O2) was calculated to be ∼0.70 V for the Au₂₅(SePh)₁₈ NCs and ∼0.30 V for the Au₂₅(SC₂H₄Ph)₁₈ NCs.⁶¹ This difference was explained based on the different dielectric environments experienced by both the cluster–cores owing to the different templates used. Thus again, the role played by a ligand in controlling the electrochemical properties of a NC is exemplified.

Fig. 11 Square-wave voltammograms (SWVs) of (A) Au₂₅(SePh)₁₈ and (B) Au₂₅(SCH₂CH₂Ph)₁₈ NCs. The experiment was carried out in CH₂Cl₂ containing 0.1 M Bu₄NPF₆ having a concentration of NCs as 3 mg mL⁻¹. Here the asterisk (*) indicates the wave for incompletely removed O₂. Reproduced with permission from ref. 61. Copyright 2014, The Royal Society of Chemistry.

So far, we have discussed how NCs can be prepared using relatively large molecules as templates. However, the situation becomes far more challenging when small molecules are used instead to prepare stable NCs. Although difficult in terms of synthetic protocols, and many-a-times stability after formation becomes an issue when such small molecules are used, recently, this topic has garnered enough scientific interest.^101,120 We have already discussed that the small moiety of large molecules may be responsible for the formation of NCs, such as the Tyr molecules present inside the serum albumin proteins act as a reducing agent of metal ions.^{63,107,109,114} Thus, to understand the role of small ligands and the subsequent fundamental aspects related to the preparation of NCs, in two different independent studies, we have recently investigated the role played by the common template, Tyr, in the generation of AgNCs and CuNCs.^96,110 For both cases, we have developed optimized and easy one-pot synthetic protocols. We have thoroughly investigated the role of Tyr in the formation of stable CuNCs using various experimental techniques as well as theoretical approaches.¹¹⁰ The as-prepared CuNCs displayed blue luminescence (at 415 nm) upon excitation at 315 nm. The spectroscopic signatures of these CuNCs were in excellent agreement with those reported for the other CuNCs prepared using the large molecule, HSA, as the template.^76,87 This observation supported the argument/conjecture regarding the role of Tyr towards the formation of CuNCs. However, the luminescence signal obtained from NCs when HSA was used as a template was far more intense than what was observed when Tyr was used instead (Fig. 12).^109,110 Additionally, the cluster–core comprised 12 Cu atoms (using HSA as a template)¹⁰⁹ against 9 Cu atoms (when Tyr was used as the template).¹¹⁰ These minor discrepancies highlight the fact that the macromolecular structure of HSA provided extra surface stability and compactness in contrast to the small molecule, Tyr. Hence, it can be rationalized that besides the role played in the formation of NCs, large molecules can provide extra surface stability to the cluster–core.

Fig. 12 Excitation-dependent emission profiles of (A) HSA-capped CuNCs and (B) Tyr-capped CuNCs. The greater luminescence intensities obtained for HSA-capped CuNCs can be readily seen from the figures. Reproduced with permission from ref. 109 and 110. Copyright 2015 and 2021, American Chemical Society.

Although only Tyr can lead to the formation of NCs, it must be mentioned that such systems are not devoid of heterogeneity. When HSA or BSA is used for the generation of NCs, generally, a narrow distribution in terms of cluster size is obtained.^63,109,114 However, when we synthesized CuNCs using Tyr as the template, a Tamarix dioica leaf-like fibrillar pattern was observed from the TEM imaging experiments (Fig. 13A).¹¹⁰ Based on several control experiments, we concluded that the origin of fibrillar-like structures was the Tyr molecule itself (Fig. 13B). The TEM images displayed that the as-synthesized system consists of an aggregated fibrillar structure along with tiny CuNCs.¹¹⁰ It is already reported that in an alkaline medium, Tyr exists as a di-Tyr molecule.^121,122 Our results suggested that during synthesis, the formation of di-Tyr is very rapid in nature (as compared to the formation of CuNCs) as the pH of the reaction medium was alkaline. This rapid formation of di-Tyr in an alkaline medium was well-corroborated by our UV-Vis absorption kinetics study.¹¹⁰

Fig. 13 (A): (i–iii) TEM images of the fibrillar-like pattern formed by Tyr ligands during the preparation of Tyr-templated CuNCs. These fibrillar structures were formed along with smaller sized CuNCs. (B): (iv–vi) TEM images displaying the aggregated Tyr molecules (due to the formation of di-Tyr) in the presence of NaOH and in the absence of Cu²⁺. Reproduced with permission from ref. 110. Copyright 2021, American Chemical Society.

The associated thermodynamics due to this transformation (from Tyr to di-Tyr) was also studied using the isothermal titration calorimetry (ITC) technique. From the ITC results, we obtained the evidence of di-Tyr formation in the presence of the added alkali (NaOH), along with the generation of hydrophobic interactions (characterized by ΔH > 0, ΔS > 0).^110,123 To support this result, we have also performed molecular docking studies and found that the distance and angle between two π clouds satisfied the pre-requisites for π–π interactions (distance: <5 Å and angle: <45°) (Fig. 14A–D).¹²⁴ It is also well known that this hydrophobic interaction is one of the major factors for fibril formation.¹¹⁰ Thus, we concluded that the π–π interaction of the Tyr or di-Tyr molecules in an alkaline medium facilitated fibrillar-like structure formation during the synthesis of CuNCs. Su and co-workers also reported the presence of di-Tyr inside the BSA molecule after the preparation of AuNCs (Fig. 14E).¹²² Although the di-Tyr molecules were also present within the system, the bulky nature of the BSA molecule provided the necessary encapsulation to AuNCs once formed, and hence, the aspect of heterogeneity has not been reported.¹²² Using the same template Tyr, we have observed a different type of heterogeneity in the case of AgNCs.⁹⁶ The time required for the formation of AgNCs was less than that required for CuNCs. Interestingly, in this study, besides the stable Tyr-templated AgNCs, the system was having larger-sized nanoparticles which were non-luminescent.⁹⁶ Once smaller-sized NCs were formed within a very short period, they were not protected instantly by the small template and hence self-aggregated to form larger-sized nanoparticles. Thus, the role played by a ligand is again established as in this case we obtained a dual-component system using a small molecule as a template.⁷⁶

Fig. 14 (A) and (C) The best-docked structures displaying the distance of one Tyr molecule bound to another Tyr molecule and one di-Tyr molecule interacting with another di-Tyr molecule, respectively. (B) and (D) The best-docked structures demonstrating the angle of Tyr bound to another Tyr molecule and di-Tyr interacting with another di-Tyr molecule, respectively. All these data suffice the pre-requisites for obtaining π–π interactions present in these systems. Reproduced with permission from ref. 110. Copyright 2021, American Chemical Society. (E) Schematic representation of the formation of di-Tyr residues inside the protein matrix. Step (i) represents the protein oxidation via the formation of AuNCs inside the protein scaffolds. Step (ii) represents the subsequent removal of AuNCs by the cysteamine-induced etching process, thereby leading to the luminescence properties of di-Tyr. Reproduced with permission from ref. 122. Copyright 2015, American Chemical Society.

The effect of surface rigidity on the luminescence properties of AuNCs was demonstrated by Pyo et al.¹²⁵ In this study, the surface of the already prepared AuNCs using glutathione as a template was more rigidified by using tetraoctylammonium (TOA) cations (Fig. 15A(i–ii)). This led to further enhancement of the QY of glutathione-capped AuNCs as compared to what was observed without the addition of TOA (Fig. 15A(iii)).¹²⁵ Similarly, by monitoring the luminescence intensities of HSA-templated AuNCs, we observed that with a decrease in temperature, the luminescence intensity of AuNCs increases and vice-versa (Fig. 15B(i)).⁶³ Upon decreasing the temperature, the protein achieves thermal compactness, and hence, the wrapping of the ligand around the cluster–core gets augmented. A blue shift in the emission maximum, which reflects a higher order of hydrophobic environment in and around the fluorophore (here AuNCs), was also observed (Fig. 15B(ii)), which further delineates our temperature-dependent observation.⁶³

Fig. 15 (A): (i) Schematic illustration of the binding of TOA to Au₂₂(SG)₁₈ NCs (color label: Au = blue; S = green). (ii) Photographic images of Au₂₂(SG)₁₈ in water and TOA–Au₂₂ NCs in toluene when viewed under a 365 nm irradiation. (iii) Luminescence spectral profiles of Au₂₂(SG)₁₈ NCs in water and TOA–Au₂₂ in toluene. The luminescence intensities have been compared with a standard fluorophore, Rhodamine B (RhB, QY = 31%), with the same optical density. Reproduced with permission from ref. 125. Copyright 2015, American Chemical Society (B): (i) Variation in the maximum luminescence intensity as a function of temperature for the HSA-templated AuNCs, as marked in the figure. (ii) Variation in the emission wavelength maximum as a function of temperature for the HSA-templated AuNCs, as marked in the figure. Reproduced with permission from ref. 63. Copyright 2021, The Royal Society of Chemistry.

Owing to their photostable and biocompatible nature, luminescent metal NCs are widely used for bio-marking studies.^62,63,67,126 For targeting the point of interest within a cell, intercellular localization studies are extremely important. Convertible organelle-targeting properties can be achieved by a controlled design of ligand exchange method.¹²⁷ Recently, Yang et al. efficiently demonstrated such a ligand exchange strategy that empowers AuNCs to specifically target mitochondria instead of lysosomes which were initially targeted prior to ligand exchange.⁷⁵ This study demonstrated that hydrosoluble glutathione (–SG) templated Au₁₈SG₁₄ NCs can accumulate mainly in lysosomes inside the cell lines. Using a suitable ligand exchange engineering, Au₁₈SG₁₂MTPB₂ NCs were prepared (where MTPB = (4-mercaptobutyl)triphenylphosphonium bromide).⁷⁵ Interestingly, these Au₁₈SG₁₂MTPB₂ NCs were more inclined to encapsulate in the mitochondrial site within the cell lines investigated.⁷⁵ Additionally, it was further demonstrated that upon photoexcitation at 638 nm, the Au₁₈SG₁₂MTPB₂ NCs can induce reduced cytotoxicity (due to the generation of lesser singlet oxygen, ¹O₂) compared to Au₁₈SG₁₄ NCs (Scheme 5).

Scheme 5 Switching of intracellular targeting organelles (from lysosomes to mitochondria) by Au₁₈SG₁₂MTPB₂ NCs and demonstration of photodynamic therapy (PDT) toward cancer cells. Reproduced with permission from ref. 75. Copyright 2018, The Royal Society of Chemistry.

Sensing

Due to their high QYs, organic dyes and quantum dots (QDs) were the most popular fluorescence probes for sensing applications.^128–136 However, tedious synthetic protocols, environmentally hazardous nature, low biocompatibility, poor water solubility, lack of cost-effective production and, most importantly, toxicity limit their usage and hence alternative solutions to circumvent these limitations are surely needed. Hence, owing to their remarkable properties as discussed earlier, luminescent metal NCs are widely used for sensing purposes.^{62,63,79–81} These NCs, which thereby serve as next-generation fluorophores, have been utilized for sensing applications in various inter-disciplinary research fields like in vitro and in vivo sensing of cations, anions, pH, temperature, viscosity, biomolecules, etc.^{19,63,67,80,86,106}

Inorganic ion sensing

From environmental pollution to industrial purposes, the detection of ions (especially metal ions) is of utmost importance as it has direct relevance to health issues. Although some instrumental techniques (e.g., ICP-OES, ICP-MS, AAS, AFS, and some X-ray based techniques) are being used extensively for the detection of elements, such approaches are not user-friendly because of the long analysis period, high power consumption, expensive instrumentation, complex analysis procedures, need for large-scale samples, limitation in in vivo detection, etc.^137,138 In this context, metal NCs become a natural choice, especially for the detection of ions based on their luminescent signatures. Metal NCs serve as optical sensors and can be categorized as “turn-on” and “turn-off” luminescence probes. The efficiency of detection, which is normally quantified from the limit of detection (LOD), categorizes these NCs accordingly.

Xie et al. first reported the detection of Hg²⁺ ions in solution using red emissive AuNCs.¹³⁹ Recently, we have reported HSA templated AuNCs as a selective sensor for Hg²⁺ ions with very high sensitivity.⁶³ These AuNCs were robust in nature against thermal and chemical perturbations except for Hg²⁺ ions, whereby the luminescence intensity gets severely quenched in the presence of these Hg²⁺ ions (Fig. 16A).⁶³ The optimum QY (>10%) and very high photostability (the AuNCs were stable more than a year) make these NCs suitable sensors (LOD ∼ 1 nM). This is the first ever approach to sense Hg²⁺ ions with HSA-templated AuNCs using the FCS technique.⁶³ This single molecular study demonstrated the ultra-sensitive nature of AuNCs towards Hg²⁺ detection with an extremely low LOD value (∼0.01 nM) which is much lower than the international standards set for Hg²⁺ levels in drinking water (10 nM) according to the U.S. EPA.¹⁴⁰ We also studied the effect of interaction of AuNCs with Hg²⁺ when this ion was bound to a biomolecule, insulin. To this effect, we have fabricated a model system by mixing insulin and Hg²⁺ forming the insulin–Hg (I–H) complex. We have concluded that these AuNCs can also sense Hg²⁺ with almost similar efficiency as observed in an aqueous medium.⁶³ From our FCS investigations, the step-wise decrement of correlation signal and the diffusion time were demonstrated which correlate our observation based on the quenching of luminescence intensity owing to the gradual destruction of the cluster–core (Fig. 16B). The selectivity toward Hg²⁺ ion detection and the visible color change of the AuNCs upon interaction with the Hg²⁺ ions are represented in Fig. 16C and D, respectively. This interaction of AuNCs and Hg²⁺ can be rationalized by the concept of metallophillic interaction (d¹⁰–d¹⁰) phenomenon.^63,139,141 Also, the higher reduction potential of Hg²⁺/Hg⁰ (E_red = 0.86 V) as compared to other heavy metals was the important factor that led to the interaction of Hg²⁺ with Au cluster–cores. Most interestingly, bio-imaging study as a function of time showed that these AuNCs can also sense the Hg²⁺ ions inside the breast cancer cell lines (MDA-MB-231) (Fig. 16E).⁶³

Fig. 16 (A) Modulations in the emission spectra of the AuNCs with the increasing concentrations of Hg²⁺. The gradual quenching of the red emission peak (λ_em = 660 nm) is due to the step-wise dissociation of the parent NC and the simultaneous generation of blue-emitting NCs (λ_em ∼500 nm) of smaller size observed at the higher concentrations of Hg²⁺ ions. (B) Auto correlation curves obtained from the FCS measurements for AuNCs with the increasing concentrations of Hg²⁺ ions. (C) Histogram displaying the selectivity of these AuNCs in sensing Hg²⁺ ions among the several other ions which were also screened. (D) Photographic images displaying the change in color of the AuNC solutions in the presence of increasing concentrations of Hg²⁺ ions, when viewed under ordinary and UV light. (E) Confocal images of the MDA-MB-231 (breast cancer) cell lines when incubated with Hg²⁺ ions in the presence of AuNCs. Reproduced with permission from ref. 63. Copyright 2021, The Royal Society of Chemistry.

In another work from our group, we demonstrated the photo-switch nature of HSA templated Ag₉:HSA NCs for the label-free detection of Co²⁺ and Zn²⁺ ions in the aqueous phase.⁹⁹ Ag₉:HSA NCs showed a “turn-off’ signal in the presence of Co²⁺ ions; the luminescence intensity of Ag₉:HSA NCs was reduced by 74% in the presence of 1 mM Co²⁺ ions (Fig. 17A). The LOD was calculated to be 100 nM. Most interestingly, these quenched NCs regained their luminescence intensity, i.e., exhibited a “turn-on” signal in the presence of Zn²⁺ ions, characterized by a LOD of 50 nM (Fig. 17A).⁹⁹ This sensing mechanism of these Ag₉:HSA NCs based on “turn-on” and “turn-off” luminescence intensities make them behave as a label-free photoswitch (Fig. 17B) which can be used to detect pollutants in water from several sources.⁹⁹

Fig. 17 (A) Histograms demonstrating the selectivity of Ag₉:HSA NCs in detecting Co²⁺ and Zn²⁺ ions in solution by “turn-off” and “turn-on” signals, respectively. (B) Schematic illustration of the mechanism of “on” and “off” luminescence features of the Ag₉:HSA NCs which make them behave as a label-free photoswitch. Reproduced with permission from ref. 99. Copyright 2014, The American Chemical Society.

He and co-workers developed thymine-rich (T-rich) DNA strand templated yellow luminating CuNCs which can efficiently detect Mn²⁺ ions in the aqueous phase by the “turn-on” signal with a detection limit of 10 μM.¹⁴² In an earlier study, Lan et al. demonstrated an interesting application of DNA-capped AgNCs which were used to detect Cu²⁺ ions in Montana soil (SRM 2710) and pond water with a very low LOD of 8 nM in water.¹⁰⁰ In another study having direct application in pharmaceutical industries, Kim and co-workers¹⁴³ successfully sensed the presence of Ag⁺ (as silver sulfadiazine) in a dermatological burn ointment, Silmazins®. In this study, Cytosine₁₂ (Cyt₁₂) DNA chain was used for the preparation of red luminating AgNCs which were induced to form a dimer in the presence of Ag⁺ ions (the monomeric AgNCs were anchored by a bridge formation) and the detection of Ag⁺ ions were demonstrated by a visual color change of the sample from red to green.¹⁴³

Recently, Pradeep and co-workers studied the effect of heavy metal ions on the luminescence signatures of BSA-coated Au₃₈ NCs.¹⁴⁴ This report demonstrated that these AuNCs can act as a fluorescent chemodosimeter for selectively sensing Cd²⁺ and Pb²⁺ through the formation of cluster aggregates and subsequent augmentation of luminescence intensity (Fig. 18A).¹⁴⁴ The DLS study showed that the aggregation phenomenon gets initiated beyond 500 ppb concentrations of both metal ions.¹⁴⁴ Shen et al. designed nanospheres and nanovesicles containing self-assembled Ag₆ NCs using polyethyleneimine (PEI) as a template.¹⁴⁵ They examined the sensing ability of these nano-aggregates and reported their superior sensing ability towards the detection of Al³⁺ ions (by monitoring the quenching of emission signal of AgNCs, Fig. 18B). In a seminal work, Ngeontae and co-workers reported the detection of Al³⁺ ions in solution with a LOD of 26.7 nM using the concept of “turn-on” luminescence signal of cysteamine-capped CuNCs.¹⁴⁶

Fig. 18 (A) Schematic representation demonstrating the augmentation in luminescence intensity of Au₃₈ NCs in the presence of Cd²⁺ and Pb²⁺ ions. Reproduced with permission from ref. 144. Copyright 2019, The American Chemical Society. (B) Selective detection of Al³⁺ ions by Ag₆ NCs. Reproduced with permission from ref. 145. Copyright 2018, The American Chemical Society.

Das et al. prepared a relatively small molecule, tripeptide glutathione-templated blue-emissive CuNCs, as a selective sensor for the detection of Fe³⁺ ions and estimated the LOD to be ∼25 nM.¹⁰¹ Addition of 1 mM Fe³⁺ to CuNCs resulted in an 80% reduction of the luminescence signal of the CuNCs (Fig. 19). Low concentrations of Fe³⁺ ions led to complex formation with the CuNCs through the mechanism of static quenching. However, further addition of Fe³⁺ ions resulted in a drastic decrease in the luminescence intensity involving the dynamic quenching phenomenon followed by electron transfer between the CuNCs and Fe³⁺ ions.¹⁰¹ Additionally, this work also delineated how these CuNCs can sense Fe³⁺ ions present in the methemoglobin of human blood, thereby proving how these can be used for the early detection of cyanosis.¹⁰¹

Fig. 19 Selective detection of Fe³⁺ ions in solution by glutathione-templated CuNCs. The process was characterized by a LOD of ∼25 nM. Reproduced with permission from ref. 101. Copyright 2015, The American Chemical Society.

Not only the cations, several reports also demonstrated the detection of anions by luminescent metal NCs. Chang and co-workers synthesized CuNCs using thiosalicylic acid (TA) as a capping agent.¹⁴⁷ This system acts as a dual sensing probe for the selective detection of nitrite (NO²⁻) and cyanide (CN⁻) anions and interestingly, it has been demonstrated that the sensing was controlled by the pH of the medium. An alkaline medium favours the detection of CN⁻, whereas NO²⁻ sensing was prominent only under acidic conditions.¹⁴⁷ Tai et al. utilized cysteamine stabilized CuNCs for the selective detection of I⁻ ions in the aqueous phase as well as in human urine.¹⁴⁸ Li et al. fabricated CuNCs which displayed an elevation of luminescence signal through an aggregation-induced process in the presence of S²⁻ ions with a detection limit of 42 nM.¹⁴⁹

pH, viscosity and temperature sensing

pH, viscosity and temperature play significant roles in governing the dynamics and associated structure-function inter-relations in complex biological systems.^150–152 Zhang and co-workers prepared stable CuNCs using natural silk fibroin (SF) as a ligand precursor and demonstrated the pH sensitivity and reversibility of these CuNCs displaying a linear relationship in the pH range of 6.08–10.05.¹⁵³ The pH of sample water collected from different tap waters and rivers was examined using these CuNCs, and when compared with commercial pH meters, the results obtained were in good agreement (Table 1).¹⁵³ Huang and co-workers¹⁵⁴ synthesized trypsin-templated CuNCs and reported the linear nature of sensing over a pH range of 2.02–12.14. In another study, Bonanno et al. reported that β-nicotinamide adenine dinucleotide phosphate (NADP) templated dual emissive AuNCs can efficiently sense very acidic pH (0.6–2.7).¹⁵⁵ As Helicobacter pylori (micro-organism) and several enteric pathogens favour acidic conditions and even gastric juice possesses a pH of ∼1.0 to 3.5, this investigation could be relevant for further applications in estimating the pH of such biological systems.

Table 1 pH values estimated from different water samples using SF-templated CuNCs (designated as pH_a) and compared with those recorded using a conventional pH meter (designated as pH_b). Reproduced with permission from ref. 153. Copyright 2016, The Royal Society of Chemistry

Sample		pHa	pHb	Relative error (%)
Tap water 1		7.72	7.76	−0.52
Tap water 2		7.77	7.84	−0.89
Tap water 3		7.76	7.79	−0.39
River water 1		8.06	8.00	0.75
River water 2		8.02	7.97	0.63
River water 3		8.10	8.04	0.74

---

To understand the microenvironmental viscosity, we have performed rotational and translational dynamics measurements of HSA-templated AuNCs inside two complex systems: a pluronic co-polymer hydrogel, F127 (Fig. 20A); living cervical cancerous cell lines, HeLa cells.⁸⁰ Using the conventional method,¹³² we have prepared the F127 hydrogel with the incorporation of AuNCs inside the gel network.⁸⁰ Anisotropy data revealed that the rotational time-scale inside the gel was almost similar to that of bulk water (Fig. 20B). From our translational dynamics measurements, we concluded that AuNCs were sensing ∼12 to 18 times more viscosity inside the F127 hydrogel compared to water.⁸⁰ From our FCS and time-resolved anisotropy experiments, we concluded that although the AuNCs were actually localized at the corona region of the hydrogel network, they were more exposed to the void or “water pool” region (Fig. 20A).⁸⁰ We have extended this work to more relevant applications by monitoring similar dynamics of the AuNCs inside the living HeLa cells and finally compared the results with the hydrogel system. We also concluded that the viscosity of the micro-environment of the HeLa cell lines around the localization site of AuNCs to be ∼23 cP (Fig. 20C), which is close to the value obtained inside the F127 hydrogel (∼12–18 cP).⁸⁰ Previously, Bhattacharyya and co-workers also studied the translational dynamics of BSA-coated AuNCs inside the cancerous MCF7 and non-cancerous MCF10A cell lines.⁶² Feng and co-workers, using the concept of aggregation induced emission, demonstrated the “turn-on” sensing ability with an increase of system viscosity of GSH-protected AuNCs and also used these NCs for the inter-cellular imaging of the A549 (lung cancer) cell lines.¹⁵⁶

Fig. 20 (A) Pictorial illustration of the micelle and gel network formed by a triblock copolymer, pluronic F127, and the localization of HSA-templated AuNCs inside the gel. The red spheres and the blue spheres denote the Au₂₃ and Au₉ NCs, respectively. (B) Fluorescence anisotropy decay of AuNCs in bulk water (Green), inside the F127 gel (red) and inside the HeLa cell (blue) (λ_ex = 405 nm, λ_em = 473 nm for both water and F127 gel and λ_em = 510 nm for the HeLa cell). (C) FCS trace with best fit of AuNCs incorporated inside the HeLa cell lines, λ_em = 510 nm. Reproduced with permission from ref. 80. Copyright 2020, John Wiley and Sons. (D) PL reversibility exhibited by HSA-templated CuNCs as a function of temperature. The protein unfolds upon increasing the temperature and a reverse phenomenon is observed when the temperature is ramped down. Reproduced with permission from ref. 109. Copyright 2015, The American Chemical Society.

In another study from our group, we have prepared CuNCs using HSA as a template (partially discussed earlier) and showed that these NCs can act as temperature sensors.¹⁰⁹ With an increase in temperature, the luminescence intensity of CuNCs reduced gradually and vice-versa. The elevation of temperature resulted in the unfolding of the protein around the core of CuNCs, i.e., decrease of rigidity and thus, the stability of the surface diminishes. Furthermore, abatement of temperature enabled the cluster–core surface to gain stability through the refolding of the protein and consequently, led to an increase of emission intensity of CuNCs (Fig. 20D). Most interestingly, this temperature-dependent luminescence property was reversible in nature and thus, this feature rendered these CuNCs as efficient temperature detectors (served as nano-thermometric probes).¹⁰⁹ Similarly, Tseng and co-workers demonstrated that BSA-coated AuNCs can display temperature sensing ability with reversibility in the range of 21–41 °C.¹⁵⁷ Huang et al. also observed the reversible and linear relationship between temperature and emission intensity in the range of 20–65 °C using lipoic acid-protected Au/Ag bimetallic NCs.¹⁵⁸

Sensing of biological systems

With the advancement of bio-imaging techniques and single molecular studies, the detection of biomolecular as well as biological systems became growing interest for researchers.

Chattoraj et al. reported the in-situ generation of AuNCs inside live cells by incorporating HAuCl₄ and the imidazolium-based room-temperature ionic liquid (RTIL, [pmim][Br]) conjugate inside the A549, MCF7, and HCT116 cancerous cell lines as well as WI38 and MCF10A normal cell lines (Fig. 21A).¹⁵⁹ They observed that the localization of evolved AuNCs was 20–40 times higher in cancerous cell lines as compared to the normal cell lines (Fig. 21B).¹⁵⁹ The higher concentration of GSH inside the cancerous cell lines facilitated the high number of AuNC generation. The emission signal, peaked at 490–530 nm, indicates the formation of the Au_8-13 cluster–core composition inside the cell lines. This observation is highly corroborated with our data where we got the emission signal of HSA-templated AuNCs around 510 nm after incorporation inside the MDA-MB-231 cancerous cell lines, but our AuNCs is red-luminating in nature (λ_em = 660 nm, Au₂₅) before endocytosis inside the cell lines.⁶³ In contrast, we did not get any fluorescence image after the incubation of AuNCs with normal cell lines MCF10A.⁶³ These results can thus help us in the discrimination of cell lines (cancerous and non-cancerous) by luminescent AuNCs (Fig. 21C and D). Besides this, the etching of relatively larger Au₂₅ NCs (to relatively smaller Au₁₃ NCs) during intercellular incorporation become understandable.

Fig. 21 (A) Confocal images of live lung cells stained by in situ generated Au-NCs: (i) blank A549, (ii) 200 μM [pmim][AuCl₄], (iii) 600 μM [pmim][AuCl₄] and (iv) WI38 at 600 μM [pmim][AuCl₄]. The scale bar corresponds to 4 μm. (B) Fluorescence spectra recorded by a spectrograph attached to the confocal microscope of the in situ generated AuNCs in live lung cells (λ_ex = 405 nm): A549 (green) and WI38 (black) at 600 μm [pmim][AuCl₄]. Reproduced with permission from ref. 136. Copyright 2016, John Wiley and Sons. (C) Confocal image of the MDA-MB-231 (breast cancer) cell line incubated with 30 μM AuNCs. (D) Confocal image of the MCF10A (normal breast cell) cell line treated with AuNCs. Reproduced with permission from ref. 63. Copyright 2021, The Royal Society of Chemistry.

Xie and co-workers displayed the sensing ability of nucleic acid stabilized AgNCs towards adenosine as well as the RNA of let-7a.¹⁶⁰ For both cases, the “turn-on” fluorescence signal strategy was used with the help of the photoinduced electron transfer (PET) mechanism.¹⁶⁰ Recently, Pramanik et al. utilized GSH-stabilized CuNCs to discriminate single nucleotide mismatches (SNM) in a 20 base pair long double-stranded DNA.¹⁶¹ This report demonstrated that CuNCs could differentiate between well-matched DNA sequences and sequences containing single mismatched base pairs formed by cytosine.¹⁶¹ The calorimetric and spectroscopic results revealed that tripeptide functionalized CuNCs possess a stronger binding affinity towards cytosine-thymine mismatched (CT MM) DNA. They also showed that CuNCs could be utilized as luminescent probes to monitor the molecular diffusion of SNM DNA using FCS.¹⁶¹ The equilibrium binding constants derived from the association and dissociation rate constants suggested that CuNCs interact with CT MM DNA with a ∼3 times higher binding strength than that of the WM DNA-CuNC system at single molecular resolution.¹⁶¹ Hosseini and co-workers reported the “turn-on” detection of sequence-specific microRNA-155 (miRNA-155) by DNA-RNA heteroduplex formation.¹⁶² Luminescent CuNCs have also been used for the discrimination of nucleosides therein.¹⁶² Ni and co-workers prepared CuNCs in the presence of citric acid with four different nucleosides as templates (adenosine (A), cytidine (C), guanosine (G) and thymidine (T)) and demonstrated that T failed to produce CuNCs whereas C produced most intensely luminating CuNCs, but A-templated CuNCs possessed the highest QY (1.34%) as compared to other templates.¹²⁰ In an interesting work, Bonis-O’Donnell and co-workers developed DNA-capped AgNCs for the selective detection of a neurotransmitter, dopamine, over other neurotransmitters under in vitro conditions.¹⁶³ Verma and co-workers demonstrated the specificity of 8-mercapto-9-propyladenine stabilized AuNCs towards the nuclei of several cell lines via macropinocytosis pathway encapsulation.¹⁶⁴ In a very recent review, Li et al. thoroughly discussed the role of AgNCs for being a potential sensor towards the detection of various viruses.⁷⁴ For example, Zhang et al. demonstrated the prominent detection of H5N1 gene using two DNA strands¹⁶⁵ and Shen et al. reported the possibility for the detection of Norovirus RNA by using GCC-loop containing DNA as the template.¹⁶⁶ In an important study, Han et al. demonstrated the detection of three viral DNAs (HIV, H1N1 and H5N1) using AgNCs as a sensor with the detection limits of 3.53 nM, 0.12 nM, and 3.95 nM, respectively.¹⁶⁷

Conclusions and future outlook

This review deals with the fundamental aspects of luminescent metal NCs. These small-sized metal-aggregates in their zero-valent oxidation states are non-toxic in nature and exhibit high cell viability. Thus, these can be safely used in deciphering dynamics inside biological self-assemblies.

However, the propensity of these NCs to undergo oxidation and self-aggregation to form larger nanoparticles makes the synthetic protocols and optimization processes more tedious and challenging. On the other hand, since the associated ligand covers the core of the metal NCs, they can interact with the system and thus, their various applications can be regulated.¹⁶⁸ Beside this, the stability, luminescence intensity and other photophysical properties of the metal NCs are also influenced by the compactness, surface coverage and functional groups inherently present within the ligands.¹⁶⁸ The solubility of the NCs in the solvent is highly influenced by the capping ligands. The hydrophilic ligands (having groups such as hydroxyl, carboxyl, amine, etc.) facilitate the aqueous phase solubility of the NCs. This enables the development of newer templates that can be used for the preparation of water-soluble NCs which can have varied applications, especially related to biological systems. Thus, the role of ligands and templates becomes extremely critical in the development of NCs. Herein, we have systematically highlighted the role of such ligands/templates in developing NCs which are not only stable but also have characteristic spectroscopic signatures. These ligands help in tuning the morphological aspects of these NCs and this in turn control their applications. A major section of this review deals with the sensing capabilities of these NCs. Although there is a plethora of reports that deal with the modifications of ligands used in the preparation of NCs which in turn control the application aspects, yet a more cumulative and comprehensive understanding is required. In this feature article, we have summarized how and why these can be used to detect metal ions with selectivity and unprecedented limit of detection. Metal ions which are detrimental to human health (like heavy metals, mercury, etc.) can be easily detected by these NCs. The fact that these NCs are photostable enables them to be used in single molecular investigations. We have showed how they can be used to sense the pH, viscosity and temperature of several biological systems along with their ability to discriminate cancerous and non-cancerous cell lines and specifically target certain organelles within a cellular environment.

Although the field related to NCs has seen a huge upsurge recently, more in-depth investigations are needed to realize their applications in biomedical research. The future of these next-generation fluorophores will be directed towards the development of catalysis, solar cells, safe and pure drinking water, and investigations which will have direct physiological and biomedical relevance such as targeted drug delivery and early detection of diseases.

Author contributions

SC collated all the data; SM conceptualized the review; both SC and SM wrote the entire review article.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

SC thanks IISER Bhopal for the fellowship and SM thanks SERB (CRG/2018/000760) for the financial support. SM thanks all the past and present members of the group. The support from IISER Bhopal, CIF IISER Bhopal and the Departmental TEM facility (FIST grant) is deeply acknowledged.

References

1. J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409.
2. L. A. Peyser, A. E. Vinson, A. P. Bartko and R. M. Dickson, Science, 2001, 291, 103.
3. C. T. Campbell, S. C. Parker and D. E. Starr, Science, 2002, 298, 811.
4. L. Shang, S. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401.
5. K. Zheng, V. Fung, X. Yuan, D. Jiang and J. Xie, J. Am. Chem. Soc., 2019, 141(48), 18977.
6. Z. Wu, Q. Yao, O. Jin, H. Chai, N. Ding, W. Xu, S. Zang and J. Xie, Angew. Chem., Int. Ed., 2020, 59, 9934.
7. T. Kawawaki, Y. Kataoka, M. Hirata, Y. Akinaga, R. Takahata, K. Wakamatsu, Y. Fujiki, M. Kataoka, S. Kikkawa, A. S. Alotabi, S. Hossain, D. J. Osborn, T. Teranishi, G. G. Andersson, G. F. Metha, S. Yamazoe and Y. Negishi, Angew. Chem., Int. Ed., 2021, 60, 21340.
8. H. Yu, B. Rao, W. Jiang, S. Yang and M. Zhu, Coord. Chem. Rev., 2019, 378, 595.
9. D. Bera and N. Goswami, J. Phys. Chem. Lett., 2021, 12, 9033.
10. W.-M. He, Z. Zhou, Z. Han, S. Li, Z. Zhou, L.-F. Ma and S.-Q. Zang, Angew. Chem., Int. Ed., 2021, 60, 8505.
11. M. R. Narouz, K. M. Osten, P. J. Unsworth, R. W. Y. Man, K. Salorinne, S. Takano, R. Tomihara, S. Kaappa, S. Malola, C.-T. Dinh, J. D. Padmos, K. Ayoo, P. J. Garrett, M. Nambo, J. H. Horton, E. H. Sargent, H. Häkkinen, T. Tsukuda and C. M. Crudden, Nat. Chem., 2019, 11, 419.
12. E. Khatun and T. Pradeep, ACS Omega, 2021, 6, 1.
13. K. Bhattacharyya and S. Mukherjee, Bull. Chem. Soc. Jpn., 2018, 91, 447.
14. Y. Cao, S. Malola, M. F. Matus, T. Chen, Q. Yao, R. Shi, H. Häkkinen and J. Xie, Chem, 2021, 7, 2227.
15. J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888.
16. J. Zheng, C. W. Zhang and R. M. Dickson, Phys. Rev. Lett., 2004, 93, 077402.
17. A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology, 2010, 21, 055103.
18. Q. Yao, Z. Wu, Z. Liu, Y. Lin, X. Yuan and J. Xie, Chem. Sci., 2021, 12, 99.
19. I. Zare, D. M. Chevrier, A. Cifuentes-Rius, N. Moradi, Y. Xianyu, S. Ghosh, L. Trapiella-Alfonso, Y. Tian, A. Shourangiz-Haghighi, S. Mukherjee, K. Fan and M. R. Hamblin, Mater. Today, 2021 DOI:10.1016/j.mattod.2020.10.027.
20. X.-D. Zhang, J. Chen, Z. Luo, D. Wu, X. Shen, S.-S. Song, Y.-M. Sun, P.-X. Liu, J. Zhao, S. Huo, S. Fan, F. Fan, X.-J. Liang and J. Xie, Adv. Healthcare Mater., 2014, 3, 133.
21. X. Yuan, Z. Luo, Y. Yu, Q. Yao and J. Xie, Chem. – Asian J., 2013, 8, 858.
22. S. Hasegawa, S. Takano, K. Harano and T. Tsukuda, JACS Au, 2021, 1, 660.
23. K. Koyasu and T. Tsukuda, J. Chem. Phys., 2021, 154, 140901.
24. Y. Song, H. Abroshan, J. Chai, X. Kang, H. Kim, M. Zhu and R. Jin, Chem. Mater., 2017, 29, 3055.
25. Q. Li, S. Wang, K. Kirschbaum, K. Lambright, A. Das and R. Jin, Chem. Commun., 2016, 52, 5194.
26. T. Udayabhaskararao and T. Pradeep, J. Phys. Chem. Lett., 2013, 4, 1553.
27. Y. Du, H. Sheng, D. Astruc and M. Zhu, Chem. Rev., 2020, 120, 526.
28. R. Jin, G. Li, S. Sharma, Y. Li and X. Du, Chem. Rev., 2021, 121, 567.
29. H. Haidari, Z. Kopecki, R. Bright, A. J. Cowin, S. Garg, N. Goswami and K. Vasilev, ACS Appl. Mater. Interfaces, 2020, 12, 41011.
30. M. Cao, R. Pang, Q.-Y. Wang, Z. Han, Z.-Y. Wang, X.-Y. Dong, S.-F. Li, S.-Q. Zang and T. C. W. Mak, J. Am. Chem. Soc., 2019, 141, 14505.
31. U. Kreibig and M. Vollmer, Optical Properties of Metal Clusters, Springer, Berlin, 1995.
32. Y. A. Eremin, SCATTERING|Scattering Theory, Encyclopedia of Modern Optics, 2005, 326–330.
33. Y. Tang, X. Yu, J. Xu, B. Audit and S. Zhang, Noble Metal-Metal Oxide Hybrid Nanoparticles, 2019, 431–455.
34. N. W. Ashcroft and N. D. Mermin, Solid State Physics, Holt/Rinehart & Winston, New York, 1976.
35. S. Link, A. Beeby, S. FitzGerald, M. A. El-Sayed, T. G. Schaaff and R. L. Whetten, J. Phys. Chem. B, 2002, 106, 3410.
36. C. Felix, C. Sieber, W. Harbich, J. Buttet, I. Rabin, W. Schulze and G. Ertl, Phys. Rev. Lett., 2001, 86, 2992.
37. X. Du and R. Jin, Dalton Trans., 2020, 49, 10701.
38. R. S. Dhayal, W. E. van Zyl and C. W. Liu, Dalton Trans., 2019, 48, 3531.
39. B. Nieto-Ortega and T. Bürgi, Acc. Chem. Res., 2018, 51, 2811.
40. W. A. de Heer, K. Selby, V. V. Kresin, J. Masui, M. Vollmer, A. Chatelain and W. D. Knight, Phys. Rev. Lett., 1987, 59, 1805.
41. W. A. de Heer, Rev. Mod. Phys., 1993, 65, 611.
42. R. L. Johnston, Atomic and Molecular Clusters, Taylor & Francis, London, 2002.
43. X. Kang and M. Zhu, Chem. Soc. Rev., 2019, 48, 2422.
44. X. Yuan, N. Goswami, W. Chen, Q. Yao and J. Xie, Chem. Commun., 2016, 52, 5234.
45. Z. Luo, X. Yuan, Y. Yu, Q. Zhang, D. T. Leong, J. Y. Lee and J. Xie, J. Am. Chem. Soc., 2012, 134, 16662.
46. Z. Wu, Q. Yao, S.-Q. Zang and J. Xie, Natl. Sci. Rev., 2021, 8, nwaa208.
47. Z. Wang, X. Pan, S. Qian, G. Yang, F. Du and X. Yuan, Coord. Chem. Rev., 2021, 438, 213900.
48. L. Zhu, M. Gharib, C. Becker, Y. Zeng, A. R. Ziefuß, L. Chen, A. M. Alkilany, C. Rehbock, S. Barcikowski, W. J. Parak and I. Chakraborty, J. Chem. Educ., 2020, 97, 239.
49. R. Jin, C. Zeng, M. Zhou and Y. Chen, Chem. Rev., 2016, 116, 10346.
50. Z. Liu, Z. Wu, Q. Yao, Y. Cao, O. J. H. Chai and J. Xie, Nano Today, 2021, 36, 101053.
51. Q. Yao, X. Yuan, T. Chen, D. T. Leong and J. Xie, Adv. Mater., 2018, 30, 1802751.
52. Q. Yao, T. Chen, X. Yuan and J. Xie, Acc. Chem. Res., 2018, 51, 1338.
53. X. Yuan, L. L. Chng, J. Yang and J. Y. Ying, Adv. Mater., 2020, 32, 1906063.
54. X. S. Shen, G. Z. Wang, X. Hong and W. Zhu, Phys. Chem. Chem. Phys., 2009, 11, 7450.
55. P. Born and T. Kraus, Phys. Rev. E, 2013, 87, 062313.
56. E. S. Shibu, M. A. H. Muhammed, T. Tsukuda and T. Pradeep, J. Phys. Chem. C, 2008, 112, 12168.
57. D. M. Chevrier, L. Raich, C. Rovira, A. Das, Z. Luo, Q. Yao, A. Chatt, J. Xie, R. Jin, J. Akola and P. Zhang, J. Am. Chem. Soc., 2018, 140, 15430.
58. P. A. Bartlett, B. Bauer and S. J. Singer, J. Am. Chem. Soc., 1978, 100, 5085.
59. G. H. Woehrle, M. G. Warner and J. E. Hutchison, J. Phys. Chem. B, 2002, 106, 9979.
60. Y. Yang and S. Chen, Nano Lett., 2003, 3, 75.
61. Y. Song, J. Zhong, S. Yang, S. Wang, T. Cao, J. Zhang, P. Li, D. Hu, Y. Pei and M. Zhu, Nanoscale, 2014, 6, 13977.
62. S. Chattoraj and K. Bhattacharyya, J. Phys. Chem. C, 2014, 118, 22339.
63. S. Chakraborty, A. Nandy, S. Ghosh, N. K. Das, S. Parveen, S. Datta and S. Mukherjee, Analyst, 2021, 146, 1455.
64. J. Duguid, V. A. Bloomfield, J. Benevides and G. J. Thomas, Jr., Biophys. J., 1993, 65, 1916.
65. A. Ono, S. Cao, H. Togashi, M. Tashiro, T. Fujimoto, T. Machinami, S. Oda, Y. Miyake, I. Okamotoa and Y. Tanaka, Chem. Commun., 2008, 4825.
66. C. M. Ritchie, K. R. Johnsen, J. R. Kiser, Y. Antoku, R. M. Dickson and J. T. Petty, J. Phys. Chem. C, 2007, 111, 175.
67. Y. Xiao, Z. Wu, Q. Yao and J. Xie, Aggregate, 2021, 2, 114.
68. Y. Zhang, C. Zhang, C. Xu, X. Wang, C. Liu, G. I. N. Waterhouse, Y. Wang and H. Yin, Talanta, 2019, 200, 432.
69. H. Zhang, Z. Zhao, P. R. McGonigal, R. Ye, S. Liu, J. W. Y. Lam, R. T. K. Kwok, W. Z. Yuan, J. Xie, A. L. Rogach and B. Z. Tang, Mater. Today, 2020, 32, 275.
70. M. Shellaiah and K. Sun, Chemosensors, 2017, 5, 3667.
71. I. Chakraborty and T. Pradeep, Chem. Rev., 2017, 117, 8208.
72. N. K. Das and S. Mukherjee, Phys. Sci. Rev., 2018, 3, 20170081,  DOI:10.1515/psr-2017-0081.
73. Q. Wang, S. Liu and Z. Tang, J. Mater. Chem. B, 2021, 9, 8174.
74. D. Li, H. Chen, X. Gao, X. Mei and L. Yang, ACS Sens., 2021, 6, 613.
75. Y. Yang, S. Wang, S. Chen, Y. Shen and M. Zhu, Chem. Commun., 2018, 54, 9222.
76. S. Kundu, M. Ghosh and N. Sarkar, Langmuir, 2021, 37, 9281.
77. S. Behzadi, V. Serpooshan, W. Tao, M. A. Hamaly, M. Y. Alkawareek, E. C. Dreaden, D. Brown, A. M. Alkilany, O. C. Farokhzad and M. Mahmoudi, Chem. Soc. Rev., 2017, 46, 4218.
78. T. Pan, W. Song, H. Gao, T. Li, X. Cao, S. Zhong and Y. Wan, ACS Appl. Mater. Interfaces, 2016, 8, 19217.
79. N. K. Das, S. Chakraborty, M. Mukherjee and S. Mukherjee, Chem. Phys. Chem., 2018, 19, 2218.
80. S. Chakraborty, S. Nandi, K. Bhattacharyya and S. Mukherjee, Chem. Phys. Chem., 2020, 21, 406.
81. S. Nandi, S. Ghosh and K. Bhattacharyya, J. Phys. Chem. B, 2018, 122, 3023.
82. M. Shamsipur, A. Barati and Z. Nematifar, J. Photochem. Photobio. C, 2019, 39, 76.
83. R. Ali, S. M. Saleh and S. M. Aly, Microchim. Acta, 2017, 184, 3309.
84. H. Xiong, W. Wang, J. Liang, W. Wen, X. Zhang and S. Wang, Sens. Actuators, B, 2017, 239, 988.
85. M. V. Romeo, E. Lopez-Martinez, J. Berganza-Granda, F. Goni-de-Cerio and A. L. Cortajarena, Nanoscale Adv., 2021, 3, 1331.
86. L. Zhang and E. Wang, Nano Today, 2014, 9, 132.
87. B. Wang, M. Zhao, M. Mehdi, G. Wang, P. Gao and K.-Q. Zhang, Mater. Chem. Front., 2019, 3, 1722.
88. Y. Zhang, C. Zhu, L. Zhang, C. Tan, J. Yang, B. Chen, L. Wang and H. Zhang, Small, 2015, 11, 1385.
89. R. C. Triulzi, M. Micic, S. Giordani, M. Serry, W.-A. Chiou and R. M. Leblanc, Chem. Commun., 2006, 5068.
90. C.-T. Chen, W.-J. Chen, C.-Z. Liu, L.-Y. Chang and Y.-C. Chen, Chem. Commun., 2009, 7515.
91. J. Sharma, H.-C. Yeh, H. Yoo, J. H. Werner and J. S. Martinez, Chem. Commun., 2011, 47, 2294.
92. Y. Wang, Y. Wang, F. Zhou, P. Kim and Y. Xia, Small, 2012, 8, 3769.
93. K. Zhang, K. Wang, M. Xie, X. Zhu, L. Xu, R. Yang, B. Huang and X. Zhu, Biosens. Bioelectron., 2014, 52, 124.
94. L. V. Nair, D. S. Philips, R. S. Jayasree and A. Ajayaghosh, Small, 2013, 9, 2673.
95. X. Yuan, Y. Tay, X. Dou, Z. Luo, D. T. Leong and J. Xie, Anal. Chem., 2013, 85, 1913.
96. S. Chakraborty, P. Sagarika, S. Rai, C. Sahi and S. Mukherjee, ACS Appl. Mater. Interfaces, 2021, 13, 36938.
97. T. Chen, Y. Hu, Y. Cen, X. Chu and Y. Lu, J. Am. Chem. Soc., 2013, 135, 11595.
98. A. Nain, Y.-T. Tseng, Y.-S. Lin, S.-C. Wei, R. P. Mandal, B. Unnikrishnan, C.-C. Huang, F.-G. Tseng and H.-T. Chang, Sens. Actuators, B, 2020, 321, 128539.
99. S. Ghosh, U. Anand and S. Mukherjee, Anal. Chem., 2014, 86, 3188.
100. G.-Y. Lan, C.-C. Huang and H.-T. Chang, Chem. Commun., 2010, 46, 1257.
101. N. K. Das, S. Ghosh, A. Priya, S. Datta and S. Mukherjee, J. Phys. Chem. C, 2015, 119, 24657.
102. Y. Zhang, H. Jiang and X. Wang, Anal. Chim. Acta, 2015, 870, 1.
103. C. Wanga, J. Wub, K. Jiangb, M. G. Humphrey and C. Zhang, Sens. Actuators, B, 2017, 238, 1136.
104. Y. Sun, J. Wu, C. Wang, Y. Zhao and Q. Lin, New J. Chem., 2017, 41, 5412.
105. H. Liu, G. Hong, Z. Luo, J. Chen, J. Chang, M. Gong, H. He, J. Yang, X. Yuan, L. Li, X. Mu, J. Wang, W. Mi, J. Luo, J. Xie and X.-D. Zhang, Adv. Mater., 2019, 31, 1901015.
106. X. Song, W. Zhu, X. Ge, R. Li, S. Li, X. Chen, J. Song, J. Xie, X. Chen and H. Yang, Angew. Chem., Int. Ed., 2021, 60, 1306.
107. U. Anand, S. Ghosh and S. Mukherjee, J. Phys. Chem. Lett., 2012, 3, 3605.
108. I. Chakraborty and T. Pradeep, Nanoscale, 2014, 6, 14190.
109. S. Ghosh, N. K. Das, U. Anand and S. Mukherjee, J. Phys. Chem. Lett., 2015, 6, 1293.
110. S. Chakraborty and S. Mukherjee, J. Phys. Chem. Lett., 2021, 12, 3266.
111. T. Peter Jr, All about Albumin: Biochemistry, Genetics and Medical Applications, 1996, Academic Press.
112. S. Naveenraj and S. Anandan, J. Photochem. Photobiol., C, 2013, 14, 53.
113. X. He and D. C. Carter, Nature, 1992, 358, 209.
114. Y. Xu, S. Palchoudhury, Y. Qin, T. Macher and Y. Bao, Langmuir, 2012, 28, 8767.
115. Y. Chen, T. Yang, H. Pan, Y. Yuan, L. Chen, M. Liu, K. Zhang, S. Zhang, P. Wu and J. Xu, J. Am. Chem. Soc., 2014, 136, 1686.
116. X. Liu, R. Hu, Z. Gao and N. Shao, Langmuir, 2015, 31, 5859.
117. L. Shang and S. J. Dong, Chem. Commun., 2008, 1088.
118. K. Morishita, J. L. MacLean, B. Liu, H. Jiang and J. Liu, Nanoscale, 2013, 5, 2840.
119. W. Zhong, M. Wen, J. Xu, H. Wang, L.-L. Tana and L. Shang, Chem. Commun., 2020, 56, 11414.
120. Y. Wang, T. Chen, Q. Zhuang and Y. Ni, ACS Appl. Mater. Interfaces, 2017, 9, 32135.
121. C. Giulivi, N. Traaseth and K. Davies, Amino Acids, 2003, 25, 227.
122. L. Su, T. Shu, J. Wang, Z. Zhang and X. Zhang, J. Phys. Chem. C, 2015, 119, 12065.
123. P. D. Ross and S. Subramanian, Biochemistry, 1981, 20, 3096 , and references therein.
124. Y. An, J. W. G. Bloom and S. E. Wheeler, J. Phys. Chem. B, 2015, 119, 14441.
125. K. Pyo, V. D. Thanthirige, K. Kwak, P. Pandurangan, G. Ramakrishna and D. Lee, J. Am. Chem. Soc., 2015, 137, 8244.
126. R. R. Nasaruddin, T. Chen, Q. Yao, S. Zang and J. Xie, Coord. Chem. Rev., 2021, 426, 213540.
127. G. H. Woehrle and J. E. Hutchison, Inorg. Chem., 2005, 44, 6149.
128. M. Li, T. Chen, J. J. Gooding and J. Liu, ACS Sens., 2019, 4, 1732.
129. J. Goicoechea F. J. Arregui and I. R. Matias, in Quantum Dots for Sensing, ed. F. Arregui, Sensors Based on Nanostructured Materials, Springer, Boston, MA., 2009.
130. N. Chaniotakis and R. Buiculescu, Nanosensors for Chemical and Biological Applications, Woodhead publishing, 2014, 267.
131. M. Ganesan and P. Nagaraaj, Anal. Methods, 2020, 12, 4254.
132. S. Chakraborty, S. Nandi, K. Bhattacharyya and S. Mukherjee, Chem. Phys. Chem., 2019, 20, 3221.
133. K. R. A. S. Sandanayake and I. O. Sutherland, Sens. Actuators, B, 1993, 11, 331.
134. N. K. Das, S. Ghosh, S. Jaiswal, A. Tewary and S. Mukherjee, Chem. Phys. Lett., 2017, 682, 147.
135. U. Pramanik, S. Chakraborty, K. Bhattacharyya and S. Mukherjee, Chem. Phys. Lett., 2021, 762, 138105.
136. S. Pramanik, A. Nandy, S. Chakraborty, U. Pramanik, S. Nandi and S. Mukherjee, J. Phys. Chem. Lett., 2020, 11, 2436.
137. B. Wei and L. Yang, Microchem. J., 2010, 94, 99.
138. E. Peňa-Vázquez, J. Villanueva-Alonso and P. Bermejo-Barrera, J. Anal. At. Spectrom., 2007, 22, 642.
139. J. Xie, Y. Zheng and J. Y. Ying, Chem. Commun., 2010, 46, 961.
140. L. Shang, L. Yang, F. Stockmar, R. Popescu, V. Trouillet, M. Bruns, D. Gerthsen and G. U. Nienhaus, Nanoscale, 2012, 4, 4155.
141. C. Guo and J. Irudayaraj, Anal. Chem., 2011, 83, 2883.
142. B. Han, R. Xiang, X. Hou, M. Yu, T. Peng, Y. Li and G. He, Anal. Methods, 2017, 9, 2590.
143. J. Lee, J. Park, H. H. Lee, H. Park, H. I. Kim and W. J. Kim, Biosens. Bioelectron., 2015, 68, 642.
144. J. S. Mohanty, K. Chaudhari, C. Sudhakar and T. Pradeep, J. Phys. Chem. C, 2019, 123, 28969.
145. J. Shen, Z. Wang, D. Sun, C. Xia, S. Yuan, P. Sun and X. Xin, ACS Appl. Mater. Interfaces, 2018, 10, 3955.
146. C. Boonmee, V. Promarak, T. Tuntulani and W. Ngeontae, Talanta, 2018, 178, 796.
147. J. Cang, C.-W. Wang, P.-C. Chen, Y.-J. Lin, Y.-C. Li and H.-T. Chang, Anal. Methods, 2017, 9, 5254.
148. Y.-T. Tai, T. Simon, Y.-Y. Chu and F.-H. Ko, Sens. Bio-Sens. Res., 2020, 27, 100319.
149. Z. Li, S. Guo and C. Lu, Analyst, 2015, 140, 2719.
150. E. Nader, S. Skinner, M. Romana, R. Fort, N. Lemonne, N. Guillot, A. Gauthier, S. Antoine-Jonville, C. Renoux, M.-D. Hardy-Dessources, E. Stauffer, P. Joly, Y. Bertrand and P. Connes, Front. Physiol., 2019, 10, 1329.
151. A. Cuecas, J. Cruces, J. F. Galisteo-López, X. Peng and J. M. Gonzalez, Biophys. J., 2016, 111, 875.
152. A. Curnutt, K. Smith, E. Darrow and K. B. Walters, Sci. Rep., 2020, 10, 8760.
153. G. Zhang, T. Xu, H. Du, Y. Qiao, X. Guo, L. Shi, Y. Zhang, S. Shuang, C. Donga and H. Ma, J. Mater. Chem. C, 2016, 4, 3540.
154. W. Wang, F. Leng, L. Zhan, Y. Chang, X. Xi Yang, J. Lana and C. Z. Huang, Analyst, 2014, 139, 2990.
155. A. Bonanno, I. Pérez-Herráez, E. Zaballos-García and J. Pérez-Prieto, Chem. Commun., 2020, 56, 587.
156. S. Pan, J. Zhou, W. Liu, Y. Ye, G. Chen, J. Xu, Z. Qian, J. Chen and H. Feng, Analyst, 2019, 144, 4483.
157. Y.-T. Wu, C. Shanmugam, W.-B. Tseng, M.-M. Hiseh and W.-L. Tseng, Nanoscale, 2016, 8, 11210.
158. H. Huang, H. Li, J.-J. Feng and A.-J. Wang, Sens. Actuators, B, 2016, 223, 550.
159. S. Chattoraj, Md. A. Amin, S. Mohapatra, S. Ghosh and K. Bhattacharyya, Chem. Phys. Chem., 2016, 17, 61.
160. K. Zhang, K. Wang, X. Zhu, Y. Gao and M. Xie, Chem. Commun., 2014, 50, 14221.
161. S. Pramanik, L. Khamari, S. Nandi and S. Mukherjee, J. Phys. Chem. C, 2019, 123, 29047.
162. Y.-S. Borgheia, M. Hosseinia and M. R. Ganjali, Clin. Chim. Acta, 2018, 483, 119.
163. J. T. D. Bonis-O’Donnell, A. Thakrar, J. W. Hirschberg, D. Vong, B. N. Queenan, D. K. Fygenson and S. Pennathur, ACS Chem. Neurosci., 2018, 9, 849.
164. V. Venkatesh, A. Shukla, S. Sivakumar and S. Verma, ACS Appl. Mater. Interfaces, 2014, 6, 2185.
165. Y. Zhang, F. Mu, Y. Duan, Q. Li, Y. Pan, H. Du, P. He, X. Shen, Z. Luo, C. Zhu and L. Wang, ACS Appl. Mater. Interfaces, 2020, 12, 48357.
166. F. Shen, Y. Cheng, Y. Xie, H. Yu, W. Yao, H. W. Li, Y. Guo and H. Qian, Anal. Biochem., 2019, 583, 113365.
167. D. Han and C. Wei, Talanta, 2018, 181, 24.
168. B. Zhang, J. Chen, Y. Cao, O. J. H. Chai and J. Xie, Small, 2021, 17, 2004381.

---