Synthesis of chiral fluorescence silver nano-clusters and study on the aggregation-induced emission enhancement and chiral flip

Abstract

Synthesis and application of fluorescent and chiral nano-clusters have been concerned greatly nowadays. In this work, the silver nano-clusters (AgNCs) with fluorescence and chirality were synthesized with solid-phase synthesis method, using racemic glutathione (GSH) as ligand. The synthesis conditions were optimized. In the process of synthesis, we were surprised to discover the aggregation-induced emission enhancement (AIEE) and chiral flip of AgNCs. The mechanism of AIEE and chiral flip were studied with high resolution transmission electron microscopy (HR-TEM), electrospray ionization-mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), fluorescence (FL) and circular dichroism (CD) spectrometry. What we found was that the hydrogen bond between GSH molecules on the surface of AgNCs induced cross-coupling agglomeration of the clusters, resulting in AIEE and chiral flip. In this study, the proposed AgNCs are expected to be used as a chiral fluorescent probe for the chiral recognition of biological and pharmaceutical molecules, and the synthesis method of chiral fluorescent AgNCs and mechanism explained here play an important guiding role in the future development of metal nano-clusters.

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Introduction

Metal nano-clusters (MNCs) represent the missing link between metal atoms (exhibiting distinct optical properties) and nanoparticles (NPs) (exhibiting plasmons properties), and display molecule-like behaviour.¹ It has been actively investigated in a variety of fields including Raman analysis, electrochemical analysis, fluorescent analysis, energy for receptor built, single molecular imaging, cell mark, organization and the living imaging, transit gene, drug delivery and cancer therapy, etc.^2–10 In recent years, many methods have been reported for the synthesis of MNCs. The basic principle of most methods is that the metal ions in the aqueous solution are adsorbed on a specific template (such as polyamidoamine dendrimers, amino acids, peptides, protein, DNA and RNA etc.), and then reduced by chemical reducing agent (such as sodium borohydride and ascorbic acid etc.) or light (including visible and UV light).^11–17 However, because MNCs with ultra-small size are very easy to reunite and gradually grow into large-size nanoparticles to reduce the large surface energy, many studies show that the synthesis of MNCs is always difficult in the aqueous solution. In addition, some other factors have a great influence on the successful synthesis of nano-clusters, such as temperature, type and quantity of reductant, template molecule and the ratio to metal ions and so on.¹⁸ So far, the general guidelines for the synthesis of MNCs seem to have not been found, and little has been known on the formation mechanism of MNCs. Compared with gold nano-clusters (AuNCs), the stability of AgNCs is more easily affected by many factors. Except for their vulnerability to atmospheric oxygen and other electron acceptors in solution, reduced silver atoms have a strong tendency to agglomerate to form large silver nanoparticle in solutions.¹⁹ So the most important thing when synthesizing stable AgNCs is to prevent aggregation. Fortunately, solid-phase synthesis is different from the traditional preparation methods (e.g., solution phase synthesis). The reduction occurs slowly and the kinetics of the reaction could be controlled well when using solid-phase reactants. So the aggregation of AgNCs to AgNPs can be avoided effectively.

Fluorescence is one of the important properties of metallic nano clusters. The size of MNCs is close to the Fermi wavelength of electrons, between metal atoms and nanoparticles, resulting in molecule-like properties including discrete energy levels, size-dependent fluorescence, good photo-stability and biocompatibility. These excellent properties make them become ideal fluorescent probes for biological application.¹⁹ Recently, a lot of research discovers that MNCs generate outstanding fluorescence by AIEE.²⁰ At present, the mainly understanding of the mechanism for AIEE is that aggregation restricts on the molecular vibrations and rotations, which reduces the energy loss through the non-radiative relaxation channel thence strikingly enhances the emission. Many factors (such as pH, incubation time and solvent etc.) can affect AIEE of nanoparticle, but little related research is reported. What is more, the origin of fluorescence is a fundamental issue, but it is not known clearly. The fluorescence may derive from the metal core (quantum size effect) or from the interaction between the metal core and surface ligands.²¹ Specifically, the fluorescence could be affected by surface ligands in one of two different ways: ligand-to-metal charge transfer (LMCT) or ligand-to-metal–metal charge transfer (LMMCT).^22,23 However, more research is needed to prove the controversy.

In addition to the fluorescence characteristics of MNCs, the chirality of MNCs has caused extensive concern for researchers in recent years. The chiral MNCs can provide sensitive optical signals, and can be used for chiral recognition. So it is extremely potential that the chiral MNCs could become a highly sensitive and selective probe, and play a significant role in the field of biological analysis. At the same time, the synthesis of chiral MNCs provides the possibility for the study of nanoscale chiral effects, and may provide important inspiration for the study of the origin of the chiral molecules in nature. At present, most chiral MNCs are prepared by inducing with chiral ligands, such as chiral thiol, DNA template, amino acid and its derivatives, polymers and so on.^24,25 However, the synthesis process need to be further improved. And few racemate is reported to be used as ligand for chiral MNCs synthesis. Although the mechanism of chiral generation has been studied and a lot of researchers have put forward different views, it still needs further confirmation and perfection. Therefore, it is very important and urgent to further explore the synthesis methods of chiral MNCs and the mechanism of chiral generation.

In this work, we referenced and improved the T. Udayabhaskararao synthetic route.²⁶ AgNCs with fluorescence and chirality were successfully synthesized, using solid phase reduction method with inexpensive racemic GSH as the template molecule. When the freshly-prepared AgNCs powder was dissolved in aqueous solution and placed at room temperature, the fluorescence would be significantly enhanced, and the CD signal would change. Through the study on the structure of AgNCs by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectrometer (FTIR), MALDI-TOF-MS and HR-TEM etc., the results showed that the main reason for the fluorescence enhancement and the chiral change was the cross-linking agglomeration of the ligand molecules on the surface of AgNCs through hydrogen bonds. The maximum fluorescence emission peak of synthesized AgNCs was located at 440 nm, and the fluorescence was very stable, which indicated that it had a great potential application in cell labeling and imaging. Meanwhile, AgNCs, which had strong circular dichroism signal, were expected to be used in chiral recognition, especially chiral drugs identification, chiral switch and chiral catalysis, etc.

Experimental

Materials

All chemicals were analytical reagent (AR) and used without further purification. Silver nitrate (AgNO₃) was purchased from Guangdong Huada Chemical Reagent Co., Ltd. (Guangdong, China). Sodium borohydride (NaBH₄) was purchased from Chengdu Apotheker Chemical Reagent Co., Ltd (Chengdu, China). Racemic GSH was bought from Sangon Biotechnology Co., Ltd (Shanghai, China). Sodium hydroxide (NaOH) was purchased from Xi'an Chemical Works (Xi'an, China). Petroleum ether (bp 60–90 °C) and n-butanol (C₄H₁₀O) were obtained from Chengdu Kelong Chemical Reagent Company (Chengdu, China). Ethylene glycol ((CH₂OH)₂), dimethyl sulfoxide (DMSO), ethanol (C₂H₆O) and acetic acid (CH₃COOH) were purchased from Chuandong Chemical Reagent Co., Ltd. (Chongqing, China). Ultrapure water (18.2 MΩ) was used throughout the experiment. All glassware and mortar were washed with aqua regia, rinsed with ethanol and ultrapure water, and dried in an oven before use.

Synthesis of AgNCs

AgNCs were prepared according to a reported procedure with some modification.²⁶ Generally, 23 mg of solid AgNO₃ (s) was added to 200 mg of racemic GSH (s) at 27 °C, and the mixture was grounded well in a mortar. 10 minutes later, 25 mg NaBH₄ (s) was added and grinded continually for 10 minutes. After that, 13 mL of ultrapure water was added slowly which resulted in the formation of a reddish brown solution. The solution was precipitated immediately by the addition of 18 mL ethanol and then the supernatant was centrifugated under 8000 rpm for about 5 minutes. After that, the precipitate was ultrasonically dissolved with ethanol and remove supernatant, which were repeated for three times. Finally, the precipitate was dried by vacuum dryer and collected as reddish brown powder. Put 12 mg of newly prepared AgNCs powders into an EP tube, and then add 4 mL of ultrapure water. The EP tube was placed in an ultrasonic instrument to dissolve for 2 minutes. The solution was placed at room temperature for 24 hours, and the solution color was changed from the dark red-brown to colorless. It is called incubation process that AgNCs powders are dissolved in water and placed for 24 hours. Schematic diagram of the synthesis process of Ag NCs is shown in Fig. S1† in the revised manuscript.

Apparatus and characterization

UV-vis absorption and fluorescence spectra were recorded by a PERSEE TU-1901 UV-vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) and a PerkinElmer LS-55 fluorescence spectrometer (Perkin-Elmer, USA), respectively. The chemical compositions of AgNCs were determined by ESI-MS on a Bruker micro TOF-Q system (Bruker Daltonics, Germany). HR-TEM images of the AgNCs were taken by a Tecnai G2 F20 S-TWIN microscope (FEI, USA) operating at 200 kV. The samples were drop-cast on carbon-coated copper grids and allowed to dry under ambient conditions. XPS was determined by a Thermo Escalab 250Xi (ThermoFisher Scientific, UK). CD was recorded by JACSO J-810 circular dichroism spectropolarimeter (WELLTECH ENTERPRISES, INC., USA). Hamamatsu Quantaurus-QY Plus (Hamamatsu, Japan) was used to measure the absolute photoluminescence quantum yield (QY) of AgNCs. MALDI-TOF-MS was carried out on a SHIMADZU MALDI-7090 (Shimadzu, Japan). FTIR was measured with an GANGDONG FTIR-650 (Beijing, China). Rotatory optic measurements were performed on a Rudolph Research Analytical Autopol VI Polarimeter (Rudolph Research Analytical, USA).

Results and discussion

Characterization of AgNCs

It is shown in the UV absorption spectrum that the prepared AgNCs exhibited a sharp absorption peak at 350 nm (Fig. 1A, olive curve). In addition, the spectra did not show the characteristic plasmon absorption, which indicated that there was no formation of silver nanoparticles. The emission peak was located at 440 nm from the fluorescence spectrum and the maximum excitation wavelength was at 353 nm (Fig. 1A, blue curve). As displayed in the inset of Fig. 1A, the solution of AgNCs was colorless under daylight, while it emitted blue fluorescence under the UV light at 365 nm. Both absorption and fluorescence spectra proved that the AgNCs could be synthesized by the present method. Absolute quantum yield (QY) measurement system was developed for the determination of AgNCs. The QY was 1.2%, which was essentially in agreement with the results of gold nano-clusters reported by Venkatesh V. et al.²⁷ The prepared AgNCs solution was placed for a month at room temperature without any protection, no aggregation occurred, and the UV absorption and fluorescence intensity stayed unchanged, indicating that the fluorescent AgNCs were extremely stable (Fig. S2†). In the process of solid phase synthesis, silver-thiolate was formed first with AgNO₃ and GSH, and then silver ions were reduced after the addition of NaBH₄. GSH plays a protective role in the final formation of nano-clusters. Also, mild reduction conditions make AgNCs emit blue light.²⁸ Most scholars report that the fluorescent AgNCs excitation wavelength is larger than 400 nm.^29,30 However, the prepared AgNCs excitation wavelength was 353 nm in near-ultraviolet region, and the energy was stronger, which should be an advantage to do fluorescence imaging.

Fig. 1 Characterization of AgNCs. (A) UV-vis absorption and fluorescence, (B) circular dichroism (CD) spectra, (C) Fourier transform infrared spectroscopy (FTIR), (D) X-ray photoelectron spectroscopy (XPS), and (E) electrospray ionization mass spectrometry (ESI-MS) of AgNCs. Fluorescence spectrum was recorded at 440 nm with an excitation wavelength of 353 nm. Emission slit: 4.0, concentration: 0.375 mg mL⁻¹. Inset in (A) photographs of AgNCs under (1) daylight and (2) UV light at 365 nm. Inset in (D) magnified EI-MS of [Ag₂(SG)₁ − H]⁻ and [Ag₄(SG)₄ − 2H]²⁻.

The CD signal of AgNCs was measured with circular dichroism spectrometer. The results showed that the CD signal for GSH and AgNCs was completely different (Fig. 1B). It was clear that there was a GSH characteristic peaks in the far ultraviolet region (λ < 250 nm, Fig. 1B, black curve), which corresponded to the chiral structure of the peptide bond. However, one positive band at 285 nm and two negative bands at 240 and 350 nm were observed in the CD spectra of AgNCs (Fig. 1B, red curve). The peak at 350 nm was located right in the UV absorption peak wavelength, indicating that a new configuration is generated for AgNCs, resulting in a new CD signal. Further, it was also shown that the chirality of AgNCs was derived from the new structure, but not from GSH.^31–34 In addition, the optical rotation value of AgNCs was −0.04, which also proved that the synthesized AgNCs did have the chiral characteristics.

The structure of AgNCs was validated with FTIR, XPS and ESI-MS. The FTIR of pure GSH and the prepared AgNCs were compared to confirm the formation of GSH-protected AgNCs. The FTIR of pure GSH and the prepared AgNCs were compared to confirm the formation of GSH-protected AgNCs. A characteristic S–H stretching band at 2549 cm⁻¹ in the GSH FTIR spectra was observed (Fig. 1C black curve), while it disappeared after the formation of AgNCs (Fig. 1C green curve), which supported the proposed formation of GSH–Ag complexes through Ag–S interactions.³⁵ For FTIR of pure GSH, the strong absorption peaks at 1590–1650 cm⁻¹ and 3300–3500 cm⁻¹ should be the characteristic absorption peaks of the amino group, and the strong absorption peaks at 1700–1725 cm⁻¹ and 1389 cm⁻¹ should correspond to the characteristic absorption peaks of carbonyl group and carboxyl group, respectively. For FTIR of the prepared AgNCs, the peaks corresponding to the amino and carboxyl groups still exist. What changes a little bit is the peak shape and absorption intensity. The reason might be due to the interaction between AgNCs and GSH molecules.^36,37 The FTIR was consistent with the results reported by T. Pradeep et al.²⁶ XPS analysis was carried out to determine the oxidation state of Ag in the samples. The peaks located at 367.6 eV and 373.6 eV represented Ag 3d_5/2 and Ag 3d_3/2, indicating the formation of Ag⁰ as expected after the reduction (Fig. 1D).^38,39 Meanwhile, O, C and S elements in XPS spectra were shown in Fig. S3.† It can be concluded from the results of XPS that AgNCs ratio in silver and sulfur atom is about 1 : 1 (Table S1†).

The structure of the prepared AgNCs was estimated using ESI-MS, which is a particularly useful technique in the case of silver due to its unequivocally isotopic pattern. ESI-MS of AgNCs is shown in Fig. 1E. Two charge states for AgNCs were observed as [M − 2H]²⁻ and [M − H]⁻ (Fig. 1E, inset). There are fragment peaks for ESI-MS of AgNCs over the range of m/z 200–1400. Peaks at 306.07, 411.97, 519.86, 826.95 m/z, correspond to [SG − H]⁻, [Ag₁(SG)₁ − H]⁻, [Ag₂(SG)₁ − H]⁻, [Ag₄(SG)₄ − 2H]²⁻, respectively. It is obvious that the molecular weight difference between the fragments of about 108 and 307, just for the single Ag atom or a single molecule of GSH. [SG − H]⁻ and [Ag₁(SG)₁ − H]⁻ were more abundant observed in mass spectra of a solution of AgNCs, which illustrated that the prepared AgNCs were easy cracking for these two forms. The results of XPS showed that the ratio of Ag and S atoms in AgNCs was about 1 : 1 (Table S1†). The results of ESI-MS showed that the molecular ion peaks of AgNCs had two negative charges, and the m/z was 826.95, which is to say, the molecular weight should be 1656. So we conclude that the main composition of AgNCs is Ag₄(SG)₄.

The synthesis and stability of AgNCs were easily affected by various factors. First, AgNCs strongly tended to interact with each other and coalescence to large nanoparticles irreversibly to reduce the surface energy. Second, the nature of the scaffold was responsible for not only sizes, but also fluorescence. In order to optimize synthesis conditions, the influence of synthesis conditions on AgNCs was discussed. Fig. S4† showed the fluorescence emission spectra of AgNCs prepared with different molar ratios (R) of GSH and AgNO₃. The result showed that the fluorescence intensity gradually increased and then decreased when R was from 1 : 1 to 4 : 1. In addition, UV absorption peak of AgNCs appeared within 370–550 nm when R was 1 : 1. However, it blue shifted to 350 nm and the absorption intensity reached the maximum with R increasing to 3 : 1 (Fig. S4,† inset). We deduced that GSH could not effectively play a protective role when the amount was low, easily resulting in the formation of larger nanoparticles. And GSH with excess amount would have an impact on the fluorescence of the nano-clusters. So we chose 3 : 1 as the optimal ratio. As to the effect of temperature on synthesis, the results showed that the absorption and fluorescence intensity reached the maximum when the temperature is 27 °C (Fig. S5†). This phenomenon may reflect the effect of temperature on reaction rate and reducibility of GSH.

The type of solvents also has effect on the synthesis of AgNCs.⁴⁰ We used different solvents to dissolve the prepared AgNCs, it showed that there were strong and narrow UV absorption appearing at 350 nm and strong fluorescence emission at 440 nm, only using water as solvent (Fig. S6,† curve f). Almost no absorption or fluorescence exists when using other solvents. Compared with what Zhentao Luo et al. have reported that weakly polar solvent can induce the fluorescence enhancement of Au(0)@Au(I)-thiolate core–shell nano-clusters,⁴¹ it's obvious that there are some differences, the reason may be related to the type of nano-clusters and the synthetic methods. It is also reported that the oxygen in solvent has influence on the synthesis of nano-clusters.⁴² This experimental results showed that, no matter how much oxygen content in the solvent, the fluorescence intensity of AgNCs at 440 nm was stable (Fig. S7†).

Aggregation-induced emission enhancement and chiral flip

In the synthesis process of AgNCs, what we found is that the newly-synthesized AgNCs presented almost no fluorescence when dissolved in water, but their fluorescence was gradually enhanced in the incubation process (Fig. 2A). Moreover, fluorescence intensity for 0–12 h increases sharply, tends to be slowly for 12–24 h, and finally keeps constant 24 h later. In particular, the fluorescence emission wavelength does not change, in spite of the gradual increase in the fluorescence intensity. In addition, the changes of UV absorption were almost the same as those of fluorescence emission (Fig. S8†), that is, UV absorption intensity gradually increased, but the maximum UV absorption wavelength did not change. More interestingly, we found that CD characteristic peaks significantly changed in the incubating time after AgNCs powder dissolved in water. The positive peaks gradually changed to negative ones, while the negative peak gradually changed to positive, which did not stop until 24 hours (Fig. 2B). Compared with the Chunlei Zhang et al. reported CD spectra of AgNCs@L-GSH and AgNCs@D-GSH,²⁴ CD spectra of the AgNCs in the beginning should be similar to AgNCs@D-GSH, but it should be similar to AgNCs@L-GSH after the incubation of the AgNCs. The changes of fluorescence emission intensity, UV absorption intensity and CD spectra showed that the structure of AgNCs might have changed significantly in the incubation process of AgNCs aqueous solution.

Fig. 2 Time-dependent evolution of fluorescence and CD signals of AgNCs after dissolved in water. (A) Left: Fluorescence spectra of dissolved AgNCs at 0, 2, 4, 6, 8, 12, and 24 h, right: plots depict the fluorescence intensity as a function of the incubation time. (B) CD spectra of dissolved AgNCs at 0, 2, 4, 6, 12, and 24 h.

Fig. 3A is the time dependent Rayleigh scattering spectra of dissolved AgNCs aqueous solution form 0 h to 24 h. As the incubation time prolonged, the scattering intensity increased, suggesting that aggregation was formed. Fig. 3B is the result of the MALDI-TOF-MS of newly-synthesized AgNCs powder dissolved in water for 0 h and 24 h. The results showed that the mass spectra of the newly-synthesized AgNCs were concentrated over the range of 300–1000 m/z, which corresponded to the main components of [SG − H]⁻, [Ag₁(SG)₁ − H]⁻, [Ag₂(SG)₁ − H]⁻, [Ag₂(SG)₂ − H]⁻. Almost no mass peak appeared at more than 1000 m/z. When AgNCs aqueous solution was incubating for 24 hours, mass spectrometry peaks decreased significantly at 300–1000 m/z, but a series of new mass peaks appeared over the range of 1000–2500 m/z. Mass spectrometry results showed that larger aggregates were formed in the process of incubation of AgNCs aqueous solution. HR-TEM was used to confirm the change of size in the process of incubation. HR-TEM images of the newly-synthesized AgNCs showed that the clusters were nearly monodispersed (Fig. 3B, insets) with an average size of 1.68 nm (Fig. S9†). But 24 hours later, it was obvious that the size of the clusters increased to about 40 nm, and the clusters were cross-linked with each other to form larger aggregates. Therefore, it could be concluded that it was the aggregation effect that led to the enhancement of fluorescence (i.e. AIEE), which should be similar with what Kaiyuan Zheng et al. have reported.⁴³ Similarly, the aggregation inducing effect also led to the chirality change of AgNCs.

Fig. 3 Characterization of the aggregation of AgNCs. (A) Time dependent Rayleigh scattering spectra of dissolved AgNCs aqueous solution form 0 h to 24 h. (B) MALDI-TOF and HR-TEM (insets) of dissolved AgNCs at 0 h and 24 h.

Mechanism of aggregation-induced emission enhancement and chiral flip

According to the results of MALDI-TOF-MS, HR-TEM and Rayleigh scattering, AgNCs aggregated obviously in the process of incubation. In order to further investigate the reasons for the aggregation of AgNCs, we determined the fluorescence emission of AgNCs under different pH conditions. The results showed that fluorescence intensity was gradually decreased with the increasing pH value and decreased apparently when the pH value was more than 7 (Fig. 4A). The fluorescence emission disappeared completely when the pH value was 12. In order to study the effects of pH value, the prepared AgNCs were subjected to pH cycling between 5.20 and 11.45 using acid and base as modulators. As showed in Fig. 4B, the fluorescence switching operation could be repeated for four consecutive cycles without attenuation. A small amount of melamine was used to induce the agglomeration of AgNCs. 3.2 mL of phosphate buffer (pH = 5.0) and 0.8 mL of melamine (1 mM) mixed, and then it was added to the solution of AgNCs. The results showed that the fluorescence emission of AgNCs was stronger than that of the control group after the melamine was added (Fig. 4C). Based on the phenomenon that fluorescence intensity of AgNCs increased obviously with the decrease of pH value and the addition of melamine, the hydrogen bond interaction between carboxyl group (–COOH) and amino group (–NH₂) of GSH molecules on the surface of AgNCs should be the main reason for the agglomeration of AgNCs. There are three amino groups in the structure of melamine, with which hydrogen bonds could be formed easily. So a small amount of melamine can induce the agglomeration of AgNCs, which leads to the enhancement of fluorescence. NaOH was added to the assembled system, and the hydrogen bond would be destroyed, which would lead to the disintegration of the cross-linked structure and the dramatic decrease of fluorescence. Upon decreasing the pH value, AgNCs were transformed to a neutral form, and then the AgNCs were agglomerated through hydrogen bonds between the carboxyl groups and amino group, which resulted restriction of intramolecular rotation (RIR).⁴⁴

Fig. 4 Effects of solution pH and melamine on the fluorescence of AgNCs. (A) Fluorescence spectra of AgNCs at different solution pH. Inset: plots of fluorescence intensity of AgNCs against pH; (B) fluorescence intensity upon the cyclic switching of the AgNCs under alternating conditions of pH 5.20 and pH 11.45; (C) time dependent fluorescence intensity with and without addition of melamine.

It is reported that the cations can induce the aggregation of metal clusters.³⁰ However, there was no significant change in the intensity of fluorescence or UV absorption when Na⁺, Zn²⁺ and Cr³⁺ was added to AgNCs (Fig. S10†). The addition of Ag⁺ did not enhance the fluorescence, on the contrary, the fluorescence was decreased. The possible reason is that the interaction of Ag⁺ and GSH is stronger than that of Ag atoms. This result indicated that the structure of synthesized AgNCs in this experiment should be significantly different from what have previously reported.

Although the exact mechanisms of fluorescence emissions of MNCs are not well known,¹ the fluorescence of MNCs is generally assigned to the electronic transitions between occupied d bands and states above the Fermi level (ca. sp bands) or the electronic transitions between the highest occupied orbital and the lowest unoccupied orbital (HOMO–LUMO).⁴⁵ Different MNCs (such as Au₅, Au₈, Au₁₃, Au₂₃, and Au₃₁ clusters) could emit UV, blue, green, red and near IR fluorescence, respectively.⁴⁶ These results suggested that the fluorescence properties depended on the MNCs size and the capping or protecting ligands bonded on the MNCs surface. In our experiment, the agglomeration effect was caused by the intermolecular hydrogen bond of ligand molecules. On one hand, ligand density and ligand chain were increased. On the other hand, the agglomeration also resulted in the restriction of intramolecular motion (RIM). And eventually, the energy loss was reduced through the non-radiative rotational relaxation channel and thus the emission efficiency enhanced apparently.^47,48 Of course, because the number of silver atom of metal core did not increase after agglomeration, the maximum fluorescence emission wavelength did not significantly alter.

Up to now, the underlying mechanisms of chirality are complicated and sophisticated. Generally, the chirality of MNCs can be generated in three ways: (i) from an intrinsically chiral metal core; (ii) from the chiral arrangement of the ligands on the surface; or (iii) from chiral induction or a vicinal effect.^49–51 In this experiment, the chirality of newly-synthesized AgNCs should be derived from an intrinsically chiral metal core, but not from GSH, although it was the racemate. Cross-linking agglomeration of AgNCs occurred through hydrogen bond, so that AgNCs monomer became large in the incubation process of AgNCs aqueous solution, resulting in greatly stiffen molecular conformation and arrangements. The aggregates of different asymmetric arrangement were formed, and it also led to the change of CD signal in the end.

Detection of chiral molecules

The interesting fluorescence and CD signal indicated that it may be used as a probe for chiral molecules. Penicillamine is a medication of the chelator class, which exists in two enantiomer forms: D-penicillamine (DPA) and L-penicillamine (LPA).⁵² DPA is used for the treatment of rheumatoid arthritis,⁵³ Wilson's disease and heavy metal poisoning,^54,55 but LPA is quite toxic.⁵⁶ Thus, the development of an analytical method for the determination of DPA is of great importance. Fig. 5A showed that the fluorescence of AgNCs was obviously enhanced with the addition of DPA, but there was little effect on the fluorescence after the addition of LPA. It indicated the fluorescence of the prepared AgNCs was highly sensitive to recognition D/LPA. In addition, the fluorescence intensity of AgNCs can also be changed significantly by glucose. Fig. 5B showed that the fluorescence intensity of AgNCs was enhanced after the addition of D-glucose. However, it did not change with the addition of L-glucose. So AgNCs has great potential applications in chiral molecules recognition. The quantitative analysis of chiral molecules will be done in the following studies.

Fig. 5 Recognition of chiral molecules using the synthesized AgNCs. (A) Fluorescence spectra and intensity histogram (inset) of AgNCs (30 μg mL⁻¹) with and without addition of DPA or LPA (150 nM). (B) Fluorescence spectra and intensity histogram (inset) of AgNCs (35 μg mL⁻¹) with and without addition of D-glucose or L-glucose (395 nM) at room temperature for 2 min. Error bars were calculated from 4 measurements.

Conclusions

In summary, AgNCs with blue fluorescence and chirality were prepared with solid grinding method using racemic GSH as the ligand. The synthesis conditions were discussed in detail and the structure of the synthesized AgNCs was determined. The prepared AgNCs was used for the chiral recognition of chiral DPA/LPA and D/L-glucose. One interesting finding is that the phenomenon of AIEE and chiral flip can occur during the incubation process of AgNCs aqueous solution. A variety of methods were used to investigate the reason why AIEE and chiral flip. Hydrogen bond interaction between GSH molecules resulted in cross-linking agglomeration of AgNCs monomers to large aggregates. And the formation of larger aggregates led to the generation of new rigid structure, eventually inducing fluorescence enhancement. In addition, the chirality of AgNCs should be derived from an intrinsically chiral metal core. The aggregates of different asymmetric arrangement led to the chiral flip in the incubation process of AgNCs. Our strategy offers a simple and novel operation for the synthesis of AgNCs with fluorescence and chiral characteristics. The prepared AgNCs are expected to be used as a chiral fluorescent probe for the chiral recognition of biological and pharmaceutical molecules. Our present mechanism will provide some references for further development of the synthesis of metal nano-clusters.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (NSFC, No. 21405174).

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Footnote

† Electronic supplementary information (ESI) available: Additional figures (Fig. S1–S10). See DOI: 10.1039/c6ra22102e

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