Designing atomically precise gold nanocluster architectures with DNA-guided self-assembly and biofunctionalization approaches

Abstract

Atomically precise gold nanoclusters (AuNCs) are nanomolecular species with unique optoelectronic properties, both at the individual and assembled levels. Herein, we demonstrate the precise ligand engineering of AuNCs, enabling the controlled grafting of single-stranded oligonucleotides onto atomically defined AuNCs of different sizes—Au₁₈ and Au₂₅—which emit in the NIR-I (600–800 nm) and NIR-II (900–1300 nm) spectral windows, respectively. These biofunctionalized AuNCs, which can be considered nanomolecular building blocks, were thoroughly characterized using complementary analytical and optical techniques, including absorption and fluorescence spectroscopy, mass spectrometry, liquid chromatography, and gel electrophoresis. Through selective DNA hybridization, we successfully assembled AuNC dimers, trimers, and AuNC–dye nanosystems with high reproducibilities and yields. This work lays the foundation for the design of AuNC–DNA superstructures with potential applications in optoelectronics, sensing, and nanomedicine.

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Introduction

Atomically precise gold clusters (AuNCs) represent a promising class of nanomolecular species with a wide variety of applications in catalysis,¹ optoelectronics² and nanomedicine and notably in sensing,^3–5 imaging^6–8 and therapy.^9,10 AuNCs exhibit molecular-like properties, such as multiple absorption bands and tunable photoluminescence (PL), which are directly linked to the gold structure and the nature and coordination of ligands stabilizing the metal core. Some recent studies have also reported remarkable new optical and electronic properties of AuNCs when they were assembled either covalently or electrostatically through aggregation¹¹ or using short organic crosslinkers,¹² polymers,^13,14 metal–organic frameworks,¹⁵ or biomolecules (siRNA^16,17 and proteins). Several recent reviews have reported the advances in this emerging field of research.^18–25

However, while such nanostructures have been, to some extent, well-characterized experimentally and through theoretical models,^{11,21,25–27} there is still a lack of a strategy to design these nanostructures via a bottom-up approach, coupled with analytical methods to determine their intermediary and final stoichiometric species.

DNA nanotechnology is considered one of the most suitable options to ensure the reproducible and well-controlled assembly of nanoparticles at different scales.²⁸ For instance, metal nanoparticles (gold, silver) or nanocrystals have been attached via post-functionalization at different distances following 1D, 2D, or 3D patterns.^29–32 The ability to either attach metal particles such as AuNCs or use AuNCs functionalized with oligonucleotides (ODNs) to initiate the formation of superstructures via self-assembly therefore seems an appropriate route.

In this work, we aimed to demonstrate how the fine control over the number of ODNs through ligand-exchange reactions on two well-described atomically precise AuNCs (Au₂₅pMBA₁₈ ^33–35 and Au₁₈SG₁₄,^36,37 where pMBA is para-mercaptobenzoic acid and SG is glutathione) could enable the creation of a set of building blocks to further generate dimer and trimer nanostructures while modulating the distance between these nanomolecular species. Each step was confirmed through molecular techniques and optical and microscopy characterizations, demonstrating the high yield and selectivity of this DNA-guided approach for assembling biohybrid architectures.

Experimental section

Chemicals

Chemical products were purchased from Sigma-Aldrich (France). All oligonucleotides were purchased from Eurogentec, except for the bicyclononyne (BCN)-containing sequence, which was prepared in our laboratory via solid-phase synthesis^38,39 using the BCN phosphoramidite monomer (5′-click easy BCN II reagent from Biosearch Technologies). The thiol-dPEG₅-azido (C₈H₁₇N₃O₃S) linker was purchased from Biosynth.

Synthesis and purification of the Au₂₅(pMBA)₁₈ clusters

The clusters were prepared by a synthesis route adapted from Bertorelle et al.⁴⁰ Briefly, pMBA (5.1 mmol, 79 mg) was first dissolved in 40 mL of methanol and 4 mL of tributylamine. Then, tetrachloroauric acid trihydrate HAuCl₄ (2.54 mmol, 100 mg) was added at ambient conditions and the solution was stirred for 30 min to form a gold thiolate/tributylamine complex. Then, to induce a slow reduction of the gold, 200 mg of trimethylamine borane was added under stirring for 2 h before adding another 200 mg of the same. The solution was left stirring overnight for 24 h to achieve the formation of clusters. Precipitation of the clusters was induced by adding 1 mL of 10% NH₄OH solution and 30 mL of diethyl ether. After centrifugation, the supernatant, which contained unwanted products and residue, was removed. Another cycle of dissolution/precipitation (1 mL H₂O; 5 mL of MeOH and 20 mL of Et₂O) was then done. Purification of the Au₂₅(pMBA)₁₈ cluster was conducted by selective precipitation. The precipitate was dissolved in 5 mL of water, and then 700 mg of ammonium acetate was added, followed by 20 mL of MeOH. Under these conditions, a precipitate appears, which was then separated by centrifugation. This precipitate was essentially composed of Au₃₆(pMBA)₂₄ with good purity. Au₂₅(pMBA)₁₈ was soluble in the supernatant and could be precipitated by adding Et₂O. Two cycles of dissolution/precipitation were done to improve the purity of the end cluster, the last one using a minimum of water/MeOH precipitated by Et₂O. Finally, the pellet was dried overnight in air.

Synthesis and purification of the Au(SG) clusters

The clusters were prepared by a synthesis route adapted from Pyo et al.⁴¹ In a typical synthesis, an aqueous solution of 20 mM HAuCl₄ (12.5 mL) and 50 mM glutathione (SG, 7.5 mL) was added to 230 mL of ultrapure water in a 500 mL Erlenmeyer flask (see the ESI† for the materials and methods). The mixture was then vigorously stirred for 2 min until the yellowish solution turned cloudy. Afterwards, the pH was adjusted to 12.0 using 1 M NaOH, which caused the color of the solution to turn clear yellow. Thereafter, 0.1 mL of diluted NaBH₄ (3.5 mM) was slowly added dropwise. The reaction solution was then stirred for 30 min. During the first 15 min, the solution slowly turned orange. Finally, the pH was adjusted to 2.5 to quench the BH₄⁻ activity and the solution was stirred slowly (150 rpm) for 6 h at room temperature. The product solution was then rotary evaporated to near dryness. To isolate Au(SG) from the raw product, solvent fractionation was conducted using water–isopropanol (IPA) mixtures. Typically, the product was dissolved in 10 mL of water, and 12 mL of IPA was added to induce the precipitation of large-sized nanoclusters, which contained different species, such as Au₂₂(SG)₁₈ in addition to smaller species (Au₁₀SG₁₀, Au₁₅SG₁₃ and Au₁₈SG₁₄). The precipitate was separated by centrifugation, and more IPA (1 mL) was added to the supernatant to induce the additional precipitation of Au(SG). This process was repeated until the supernatant was clear.

Biofunctionalization of the nanoclusters by ligand-exchange reactions

In the case of ODNs, ligand exchange was performed using thio-hexyl containing ODNs (with the HS-(CH₂)₆ linker inserted at the 3′-end, 5′-end or internal position). In typical conditions, 100 μL of AuNCs (100 μM) was mixed with 200 μL of ODN-SH (100 μM), and then the triethylammoniun acetate (TEAA) concentration was adjusted to 300 mM. The reaction solution was then incubated in an Eppendorf ThermoMixer at 40 °C with stirring at 600 rpm overnight. The resulting reaction mixture was analyzed and purified through PAGE and HPLC to separate the different AuNC(n)–ODN(n) species.

Ligand exchange was also performed using a thiol-dPEG5-azido (C₈H₁₇N₃O₃S) linker. Typically, for 100 μL of AuNCs (100 μM), 200 μL of thiol-dPEG₅-azido (200 μM) diluted in a 50% acetonitrile/water solution was added. Next, TEAA (1 M) was added to reach a final concentration of 300 mM. The reaction mixture was incubated in an Eppendorf ThermoMixer at 60 °C with stirring at 600 rpm overnight. The reaction was monitored by MALDI-ToF mass spectrometry and stopped once the desired distribution of N₃ on the NC was achieved. The Au₂₅(pMBA)–N₃ mixture was then purified by precipitation and analyzed by absorbance measurements and RP-HPLC.

Biofunctionalization of the nanoclusters by click reactions

In similar conditions to those used in the ligand exchange, 100 μL of azide-modified gold nanoclusters (Au₂₅(pMBA)–N₃) (100 μM) was mixed with 200 μL of 5′-BCN-ODN (100 μM). Next, TEAA was added to reach a final concentration of 300 mM. The reaction took place at room temperature overnight.

DNA hybridization

AuNCs biofunctionalized with single-stranded oligonucleotides were hybridized with their complementary strands with or without AuNCs or dye labeling by mixing both at similar concentrations, then raising the temperature to 75 °C for 5 min and finally allowing to cool down to room temperature in 1 h.

Native polyacrylamide gel electrophoresis (PAGE)

Native PAGE separation was carried out using a vertical gel electrophoresis unit and 19 : 1 acrylamide : bis(acrylamide) ratio. The separating gels were prepared with a final concentration of 25% to separate the sub-AuNC species and 20% to separate the AuNC–ODN subspecies. In all cases, a stacking gel with 3% concentration was prepared. The elution was done using standard TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at a cool temperature. The AuNC and AuNC–ODN structures (20 μM) were dissolved in 20% (v/v) glycerol/TBE solution. The sample solutions were then loaded onto the stacking gel (25 μL per well) and eluted for 4 h at a constant voltage mode (150 V) to achieve sufficient separation. The gels were imaged either in the visible or NIR region by an ultraviolet gel documentation system (Gel Doc XR+ System, BioRad, USA) or in the SWIR optical window (900–1700 nm) according to the emission wavelength of the examined AuNCs.

NIR-II imaging

This was performed using a NIR-II Princeton camera 640ST (900–1700 nm) coupled with a laser excitation source at λ = 808 nm (100 mW cm⁻²). A short-pass excitation filter was used at 1000 nm (Thorlabs) and a long-pass emission filter (LP) set at 1064 nm (Semrock) was coupled on to a 35 mm lens (Navitar, N.A. = 4).

Reverse phase high-pressure liquid chromatography (RP-HPLC)

RP-HPLC was used to analyze and purify the different AuNCs and AuNC–ODN conjugates. Typically, the purifications were performed by reverse phase HPLC using a C18 column (Uptisphere, 250 × 4.6 mm, 5 μm, 300 A) with a gradient of acetonitrile in 10 mM triethylammonium acetate buffer. The detection was performed by UV–visible absorbance measurements using a diode array detector from a series 1100 Agilent chromatographic system. After concentration of the collected fractions under vacuum, the samples were quantified by absorbance measurements using a microvolume spectrophotometer (Denovix ds-11). Then their purity and integrity were verified using an RP-HPLC system (Agilent Technologies, 1260 Infinity) coupled with an electrospray mass spectrometer (Agilent Technologies single quadrupole 6120). Here, 200 pmol was injected on to the column (Uptisphere C18, 150 × 2 mm, 3 μm, 300 Å) and eluted with a gradient of acetonitrile in triethylammonium acetate buffer (5 mM). Peaks corresponding to the multiple-charged ions were recorded in the range of 600–1100 m/z in the negative mode and the mass was calculated with a deconvolution algorithm.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS)

The mass spectra were obtained in the negative mode on a time-of-flight Microflex mass spectrometer (Bruker, Wissembourg, France), equipped with a 337 nm nitrogen laser and pulsed delay source extraction system. The matrix was prepared by dissolving 3-hydroxypicolinic acid in 10 mM ammonium citrate buffer. A mixture of a purified DNA sample (10 pmol; 1 μL) was added to the matrix (1 μL) and spotted on a polished stainless steel target plate using the dried droplet method. The spectra were calibrated using reference oligonucleotides of known masses.

Native mass spectrometry (MS)

Native MS analysis was carried out on a quadrupole time-of-flight mass spectrometer (Q-TOF Ultima, Waters Corporation, Manchester, U.K.). Ions were generated using a nanoflow electrospray (nano-ESI) source. Nanoflow platinum-coated borosilicate electrospray capillaries were used (purchased from Thermo Electron SAS, Courtaboeuf, France). The following instrumental parameters were used: capillary voltage = 1.2–1.3 kV, cone potential = 40 V, RF lens-1 potential = 40 V, RF lens-2 potential = 1 V, aperture-1 potential = 0 V, collision energy = 30–140 V, and microchannel plate (MCP) = 1900 V. All the mass spectra were calibrated externally using a solution of cesium iodide (6 mg mL⁻¹ in 50% isopropanol) and were processed using Masslynx 4.0 software (Waters Corporation, Manchester, U.K.).

Optical measurements

All the spectroscopic measurements were performed in 1 cm quartz cuvettes (Hellma GmbH) at room temperature using air-saturated solutions. The absorption spectra of the diluted AuNC and AuNC–ODN samples in water and in TEAA buffer (10 mM) were recorded using an Agilent Cary 300 UV–vis spectrophotometer between 190 and 800 nm. Steady-state photoluminescence spectra were measured from 500 to 800 nm on a Fluoromax (Perkin Elmer) spectrofluorometer and between 600 and 1750 nm with a calibrated FSP 920 spectrofluorometer (Edinburgh Instruments, Edinburgh, United Kingdom) equipped with a nitrogen-cooled PMT R5509P. Determination of the PL emission enhancement was estimated using the area under each curve using GraphPad Prism software.

Transmission electron microscopy (TEM)

Around 3 μL of AuNC solution was deposited onto an ultrathin carbon film or graphene support film. Imaging was performed on a Thermo Fisher Tecnai F20 TEM system operating at 200 kV. Data were collected on a Ceta detector (4k × 4k).

High-resolution transmission electron microscopy (HRTEM)

The metal core sizes were determined by HRTEM with a 200 kV monochromated TEM instrument using dispersed Au NCs on ultrafine carbon films.

Scanning transmission electron microscopy (STEM)

The nanostructures were imaged with the STEM mode using a Zeiss Merlin microscope. Images were collected using both bright field and dark field detectors at 30 kV with a beam current of 250 pA with magnifications between 2× and 200k×.

List of DNA sequences used in this study

The structures of linkers and dye monomers are shown in the ESI (see Fig. S1†)

Name	Sequence
5′-(Thiol)-5mer	5′ (Thiol-C6)-CAC-GT 3′
17mer	5′ ACA-TTC-CTA-AGT-CTG-AA 3′
17mer complementary	5′ TTC-AGA-CTT-AGG-AAT-GT 3′
5′-(Thiol)-17mer	5′ (Thiol-C6)-ACA-TTC-CTA-AGT-CTG-AA 3′
5′-(Thiol)-17mer complementary	5′ (Thiol-C6)-TTC-AGA-CTT-AGG-AAT-GT 3′
17mer complementary-(Thiol)-3′	5′ ACA-TTC-CTA-AGT-CTG-AA-(C6-Thiol) 3′
17-mer (internal Thiol) complementary	5′ TTC-AGA-CT·(Thiol-C6)dT·AGG-AAT-GT 3′
5′-(Cy3)-17-mer	5′ (Cy3)-ACA-TTC-CTA-AGT-CTG-AA 3′
5′-(BCN)-15-mer	5′ (BCN)-TGA-ACT-GCA-GCT-CCT 3′

Results and discussion

Au₂₅(pMBA)₁₈ and Au₁₈(SG)₁₄ were synthesized following protocols established in the literature.¹ These syntheses approaches led to high yields of atomically precise AuNCs, although with some size heterogeneity, as observed by PAGE (Fig. 1A and Fig. S2A†). In the literature, several approaches have been applied to isolate a single atomically precise size with high purity, such as selective precipitation and polyacrylamide gel electrophoresis (PAGE).⁴² However, while PAGE offers high resolution and purity, it is time-consuming and requires multiple washing steps to remove acrylamide, often resulting in relatively low AuNC yields. Therefore, we opted for an alternative approach based on reverse-phase high-pressure liquid chromatography (RP-HPLC) to separate atomically precise AuNCs.

Fig. 1 (A) RP-HPLC analysis coupled with absorbance measurements for the Au₂₅pMBA₁₈ crude reaction mixture, eluted on a gradient from 0% to 20% acetonitrile in 10 mM TEAA buffer, showing different AuNC subspecies eluted at different retention times; inset shows a photograph from PAGE analysis of the same Au(pMBA) crude reaction mixture. (B) Corresponding absorbance spectra of each eluted subspecies. (C) MALDI-ToF MS spectrum of purified Au₂₅pMBA₁₈. (D) NIR-II PL spectrum of Au₂₅pMBA₁₈ dispersed in water; λ_exc. = 808 nm. (E) HRTEM image of Au₂₅pMBA₁₈.

The choice of solvent gradient is critical for the efficient separation of AuNC species and depends on the nature of the ligands stabilizing the gold core—in this case, pMBA and SG. Here, crude reaction mixtures of Au₂₅(pMBA)₁₈ (50 μL, 10 μM) were injected into an RP-HPLC system equipped with a UV–visible absorbance detector, with a linear acetonitrile gradient from 2% to 20%, in 10 mM TEAA buffer. Atomically precise AuNCs were then separated based on differences in their retention time related to their molecular weight, polarity, and hydrophobicity (Fig. 1A). The isolated AuNCs were identified according to their known absorbance signatures reported in the literature^40,43–45 and were confirmed by mass spectrometry to be respectively: Au₃₆(pMBA)₂₄, Au₂₅(pMBA)₁₈, Au₂₈(pMBA)₂₀ and Au₁₀(pMBA)₁₀ (Fig. 1B and Fig. S3†). For instance, Au₂₅(pMBA)₁₈ showed the typical absorption bands of this species at 370, 450, 480, 690, and 800 nm.³⁴

The glutathione-stabilized AuNCs were purified in a similar manner, but using a lower acetonitrile gradient than for Au(pMBA) to enable the separation and extraction of three species, namely Au₁₅(SG)₁₃, Au₁₈(SG)₁₄ and Au₂₂(SG)₁₈ (Fig. S2A and B†), with their compositions assigned based on their absorbance profiles⁴⁶ (Fig. S2C†) and ESI mass spectrometry data (data not shown).

Au₂₅(pMBA)₁₈ and Au₁₈(SG)₁₄ were chosen for the following experiments. The composition of the Au₂₅(pMBA)₁₈ was confirmed by MALDI-ToF MS measurements with a major peak at 7681.1 Da (theoretical mass = 7681.3), but with additional peaks corresponding to Au₂₅(pMBA)₁₇ and Au₂₅(pMBA)₁₆ detected (−1 or −2 pMBA) either due to mild fragmentation during ionization or due to the presence of these subspecies in the sample (Fig. 1C). These AuNCs exhibited broad photoluminescence (PL) emission in the NIR-II window under infrared excitation (λ_exc. 808 nm) (also known as shortwave infrared, SWIR), with a maximum at around 1100 nm (Fig. 1D). The HRTEM image depicted in Fig. 1E confirmed their ultrasmall size (<2 nm) and semi-crystalline structure with atomic precision.

The composition of Au₁₈(SG)₁₄ was also confirmed by native MS analysis (Fig. S2D†), with sizes smaller than 2 nm observed by HRTEM (Fig. S2E†). Au₁₈(SG)₁₄ exhibited PL in the NIR-I window with a maximum at 680 nm (λ_exc. 450 nm) (Fig. S2F†).

Oligonucleotide functionalization

To biofunctionalize atomically precise AuNCs with DNA strands, two strategies were tested: (1) azide functionalization of the AuNC surface via ligand exchange, followed by ODN binding through click chemistry, and (2) direct ligand exchange with thiol-modified ODNs on the AuNC surface.

In the first approach, Au₂₅pMBA₁₈ was initially modified with a short pegylated thiol linker (thiol-PEG₃-N₃). The reaction was stopped after 24 h in order to prevent the broad distribution of this linker on the AuNC surface from 5 to 17 PEG₃-N₃ (data not shown). MALDI-ToF mass spectrometry indicated a distribution from 1 to 4 PEG₃-N₃ ligands per AuNC (Fig. S4A†) with no changes in the absorbance spectrum (Fig. S4B†), indicating the conservation of the Au₂₅ structure.⁴⁶ RP-HPLC analysis of Au₂₅(pMBA)–N₃ showed that the azide-functionalized AuNCs were more hydrophobic and less polar due to the presence of the N₃ group, resulting in longer retention times (Fig. S4C†). Subsequently, a click reaction was carried out (see methods) with Au₂₅(pMBA)–N₃ and BCN-15mer, resulting in the successful conjugation, as observed by PAGE, which revealed three distinct new bands (Fig. S5†).

While this method provided relatively good stoichiometric control of the azide groups per AuNC, the long experimental setup (ligand exchange + click reaction) led us to pursue the second conjugation approach by direct ligand exchange with a thiol-modified ODN. By controlling the reaction stoichiometry and applying purification methods, we were able to graft a defined number of ODNs onto both Au₂₅(pMBA)₁₈ and Au₁₈(SG)₁₄. We further applied different oligonucleotides with different numbers of nucleobases (5mer and 17mer), with thiol modification on the 5′ or 3′ terminal of the ODNs or on any internal nucleobase.

By tuning the initial ratio of thiol-ODNs to AuNCs, it was possible to obtain species with a stoichiometric number of ODNs on the AuNC surface. Although RP-HPLC could not effectively resolve the individual species in the Au₂₅(pMBA)₁₈–ODN_(n) conjugates, PAGE analysis revealed three nicely distinguished bands that exhibited the NIR-II luminescence of the separated subspecies of Au₂₅(pMBA)_(18−n)-5mer_(n) (Fig. 2A). The molecular composition of each band was determined by MALDI-ToF-MS and the results confirmed the replacement of pMBA ligands by thiol-5mer ODNs with Au₂₅(pMBA)_(18−n)-5mer_(n) (n = 0, 1, 2) + 1501 + 3000 (Fig. 2B), where (+1504 m/z and 3008 m/z) correspond to the mass of one thiol-5mer subtracted by the mass of one pMBA ligand and the mass of two thiol-5mers subtracted by the mass of two pMBA ligands, respectively.

Fig. 2 (A) Native PAGE analysis of the ligand-exchange reaction mixture of Au₂₅(pMBA)₁₈ conjugated with thiol-5mer; the original gel is shown in Fig. S6.† (B) MALDI-ToF mass spectrometry of the ligand-exchange reaction of Au₂₅(pMBA)₁₈ with thiol-5mer. (C) Native PAGE analysis of Au₂₅(pMBA)₁₈ conjugated with thiol-17mer (A), its complementary strand thiol-17mer (A′), and with the thiol (mid sequence)-17mer (A′′); the original gel is shown in Fig. S7.† (D) Native ESI-MS for Au₂₅(pMBA)₁₈ grafted with a single thiol-17mer (red asterisk).

To evaluate the efficiency of conjugation with longer ODNs suitable for hybridization (15-mer minimum to provide stable hybridization with the complementary strand), a similar experiment was performed with Au₂₅(pMBA)₁₈ using a terminal thiol-modified 17-mer and its complementary sequence, as well as a 17-mer with the thiol group positioned in the middle of the sequence. The gel bands of Au₂₅(pMBA)_(18−n)-17mer_(n) revealed four bands with similar migration using either the thiol-17mer (Fig. 2C, lane A) or the complementary sequence thiol-17mer (Fig. 2C lane A′), as expected. Interestingly, four bands with slightly faster migration were observed when using the 17-mer sequence containing the thiol in the middle of the sequence (Fig. 2C lane A′′). It should be noted that almost no band corresponding to the free Au₂₅(pMBA)₁₈ was observed in column A, A′, A′′ in PAGE, suggesting the efficient ligand exchange of the thiol-ODN with the AuNCs. The first band corresponding to Au₂₅(pMBA)₁₈ with a single 17-mer (Fig. 2C, lane A) was cut and then extracted. Native ESI-MS (nano-spray) measurements in the positive mode (Fig. 2D) suggested the presence of Au₂₅(pMBA)₁₇-17mer₍₁₎ species with +3, +4 and +5 ionization charges. We could also still observe free Au₂₅(pMBA)₁₈ and Au₂₅(pMBA)₁₆-17mer₍₂₎, indicating a possible post-ligand exchange reaction, or a re-organization of the structure during the extraction of the gel due to its low stability.

These results demonstrate that Au₂₅(pMBA)_(18−n) nanoclusters with one, two, three, or four 17-mer ODNs could be obtained by adjusting the AuNC-to-ODN ratio. Although species with more than four ODNs per AuNC could be generated using different ratios, a higher number of ODNs can lead to increased heterogeneity, resulting in overlapping gel bands and reduced resolution. However, since resolving these higher-order species was not the objective of this study, they were excluded from further analysis.

To demonstrate the universality of this approach, the ligand-exchange reaction was applied to another AuNC, namely Au₁₈(SG)₁₄. A lower concentration of thiol-17mer was used compared to the experiment with Au₂₅(pMBA)₁₈ to target the formation of the monosubstituted Au₁₈(SG)₁₃-17mer₍₁₎. RP-HPLC coupled with absorbance spectroscopy was conducted to separate the different species (Fig. 3A). The top of this figure shows the RP-HPLC analysis of thiol-17mer, which was eluted at 36.4 min. The bottom of this figure shows the results for the analysis of the ligand-exchange reaction mixture, where it can be seen that the peak for the free thiol-17mer completely disappeared, indicating the complete ligand exchange between the glutathione ligand and the thiol-17mer. Two species could be isolated here: monosubstituted Au₁₈(SG)₁₃-17mer₍₁₎ (26.5 min), and Au₁₈(SG)₁₄ in excess at 9.2 min. The UV–visible absorbance spectra of these 3 species (Fig. 3B) confirmed the efficient and controlled ligand exchange on the AuNCs stabilized either by the pMBA or SG ligand.

Fig. 3 (A) Top shows the RP-HPLC analysis for thiol-17mer, while the bottom shows the RP-HPLC analysis for Au₁₈(SG)₁₄ conjugated with thiol-17mer with a linear gradient from 0% to 12% of acetonitrile in 10 mM TEAA, and detection at 260 nm. (B) Corresponding absorbance measurements of the three peaks assigned to thiol-17mer, Au₁₈(SG)₁₄, and Au₁₈(SG)₁₃-17mer₍₁₎.

DNA hybridization and control of the nanostructures

Gold nanoclusters coupled with a controlled number of oligonucleotides could be used as the building bricks for many nano-architectures. This would pave the way for numerous applications requiring precise, quantitative measurement and analysis.

As a proof of concept, an experiment was performed to control the distance between single NIR-II emitting AuNCs and an orange emitting organic dye (Cy₃). Au₂₅(pMBA)₁₇-5′-17mer₍₁₎ or 17mer₍₁₎-3′-Au₂₅(pMBA)₁₇ were hybridized with their complementary strand labeled with Cy3 dye on the 5′ end (5′-Cy3-17mer). Two possible double helix constructs were formed, with the distance between the AuNC and Cy3 being approximately 0 nm when they were on the same side or approximately 6 nm when they were on opposite sides. PAGE analysis of the two constructs and controls (5′-Cy3-17mer, Au₂₅(pMBA)₁₈, and double helix-Cy3) was performed by NIR-II imaging (Fig. 4A) and by red imaging (Fig. 4B). The double helix structures showed slower migration than the free AuNCs. Double helix-Cy3 showed only red emission with a higher molecular weight than 5′-Cy3-17mer. As expected, the two constructs with the double-helix structure AuNC–Cy3 at 0 and 6 nm distance showed similar migrations (i.e., same molecular weight), which were higher than that of the double helix-Cy3. Interestingly, while both red and NIR-II emissions were detected with AuNC–Cy3 at 6 nm, only NIR-II emission was detected for AuNC–Cy3 at the 0 nm distance, whereby the red emission was fully quenched. For instance, Pyo et al. demonstrated the ability to use Au₂₅(pMBA)₁₈ as a pH sensor by quenching the fluorescent azadioxatriangulenium dye.⁴⁷ This implies that AuNCs, like any gold nanoparticles, are good quenchers when the organic dye is located in close proximity to the AuNC surface.^47,48

Fig. 4 Native PAGE analysis of the coupling between Cy3 dye and AuNCs through DNA hybridization (17-mer). Gel imaging was recorded using an NIR-II camera (A) and a BioRad gel imager with a Cy3 filter (B). The original gel is shown in Fig. S8.†

With the library of AuNCs–ODNs in hand acting as nanomolecular building bricks, we intended to generate AuNCs dimers and trimers with high yields. We achieved this by the DNA hybridization of 17mer with AuNCs from one single ODN per AuNC and the same structure as the complementary ODNs (dimers) and from two ODNs per AuNC with a complementary single ODN per AuNC (trimers). Briefly, 5′-Au₂₅(pMBA)₁₇-17mer₍₁₎ (A) (10 μM) was hybridized with the complementary 5′-Au₂₅pMBA₁₇-17mer (A′) (10 μM) (dimers) and 5′-Au₂₅(pMBA)₁₆-17mer₂ (A′) (5 μM) was hybridized with two complementary 17mer (A′) with AuNCs on the 5′ end. The self-assembled dimer and trimer structures resulted in distances around 7 nm between the centers of the AuNCs, considering the ODN sequence. The PAGE analysis in Fig. 5A shows bands for the AuNCs alone, the AuNCs coupled with one ODN, dimers, and trimers, which were well-separated. The lower migration of the dimers and trimers, their sharp bands and the absence of unreacted 5′-Au₂₅(pMBA)₁₇-17mer₍₁₎ indicated the complete hybridization without the formation of extra species or aggregates. We then cut the bands related to the dimers and the trimers, washed them and resuspend them in water to determine their morphology and their optical properties. The TEM images in Fig. 5D suggest the presence of dispersed dimers and trimers. The observation of such structures is quite challenging, especially considering the sample preparation in the presence of salt, the low contrast of DNA and the ultrasmall size of the Au NCs. However, we were able to confirm the presence of a high number of dimers under mild conditions by STEM (Fig. S9†) despite the low resolution of the instrument. A total of 83 dimers and 14 trimers were observed in the TEM images, each with an intercluster distance of less than 7 nm, which was in agreement with the theoretical size of the double-stranded 17-mer at 5.78 nm. The absorbance spectrum of the AuNC dimers was similar to that of the single AuNCs, with the same specific absorption bands, indicating there had been no modification of the structure of the metal nanoclusters (Fig. 5B). The PL spectra showed a rather small enhancement around 1.4-fold when Au₂₅(pMBA)₁₈ had one ODN, with no significant changes observed when hybridized in dimers or trimers (Fig. 5C). This could be explained by the rather long distance (>5 nm) between AuNCs, which prevented efficient energy transfer. Moreover, this assembly was too small to collective induce an aggregation-induced fluorescence (AIE) effect,^49–51 and so the slight increase in the PL intensity might be related to the increase in the rigidity of the AuNCs in the dimers, therefore reducing the nonradiative process.^11–13

Fig. 5 (A) Native PAGE analysis of the hybridization reaction resulting in the Au₂₅(pMBA)-17mer dimer and trimer structures, image was taken using an NIR-II camera; the original gel is shown in Fig. S11.† (B) Absorbance and (C) PL spectra of dimers in comparison with free AuNCs dispersed in water; λ_exc. 808 nm. (D) TEM images of the Au₂₅(pMBA)-17mer dimer and trimer structures.

A similar experiment was performed using 5′-Au₁₈(SG)₁₃-17mer₍₁₎ and either the free complementary 17mer or when linked to the AuNCs. PAGE analysis showed the good selectivity and efficient hybridizations with NIR emitting bands at lower migration, thus validating the nanostructuring approach with a different type of AuNCs (Fig. S10†).

Conclusion

In summary, we present a ligand-exchange-based method for precisely controlling the number of various ODNs on atomically precise gold nanoclusters (AuNCs) to create nanomolecular building blocks. Several analytical techniques, notably RP-HPLC coupled with absorbance and mass spectrometry, confirmed the hybridization of the ‘AuNC–DNA blocks’ into dimeric and trimeric biohybrid structures, as well as the formation of high-purity AuNC–dye nanosystems. We anticipate that this work will pave the way for new gold nanocluster architectures with DNA-guided self-assembly, offering potential applications in optoelectronics, sensing, and nanomedicine.

Author contributions

The manuscript was written through contributions of all the authors. All the authors have given approval for the final version of the manuscript.

Data availability

All the raw data related to the physico-chemical, analytical, optical characterizations will be available upon request from the two corresponding authors: Dr Xavier Le Guével (xavier.le-guevel@univ-grenoble-alpes.fr) and Dr Didier Gasparutto (didier.gasparutto@cea.fr).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research project is financially supported by the Grenoble Alpes University (CBH-EUR-GS, ANR-17-EURE-0003 and Labex ARCANE, ANR-11-LABX-003) and the French Agence Nationale de la Recherche (project SIREN-ANR-20-CE92-0039, project NanoGold – ANR-22-CE29-0022). D. G. is grateful to the French Alternative Energies and Atomic Energy Commission (CEA PTC-PE research program, MultiMASS project) that supported part of this study. H. K. acknowledges the financial support of the Cross-Disciplinary Program on Instrumentation and Detection of CEA, the French Alternative Energies and Atomic Energy Commission (CEA PTC-ID LCTEM). This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB) supported by FRISBI (ANR-10-INBS-05-02) and Labex GRAL (CBH-EUR-GS, ANR-17-EURE-0003). The IBS Electron Microscope facility is supported by the Auvergne–Rhône–Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS IBiSA. The authors would like to thank Wai Li Ling and Laetitia Rapenne for the TEM and HRTEM characterizations.

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Footnote

† Electronic supplementary information (ESI) available: The physico-chemical and optical characterization of AuSG (S2), AupMBA (S3), AupMBA-N3 (S4) as well as PAGE and microscopy measurements of the AuNC-ODNs and AuNC dimer structures (S5–S11). See DOI: https://doi.org/10.1039/d5nr00905g

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