Introduction

Amongst the plethora of different shapes and chemistries, two-dimensional nanomaterials are of particular interest because of their anisotropic shape and exceptional optical, catalytic, magnetic and mechanical properties, either as single nanocrystals or self-assemblies1,2. Furthermore, such materials can serve as the base for the introduction of chirality, even for materials exhibiting face-centered cubic crystal structure symmetry and plasmonic response, such as noble metals3. The union of plasmonic and chiroptical responses present some intriguing opportunities, and many approaches have already been developed4, especially on gold5. In fact, early examples from the 1950s6, which followed theoretical work on screw dislocations7,8, described gold nanoparticles with nanoplate screw formation in aqueous solutions. Interestingly, an unusually high Burgers vector above 10 nm was also reported, speculating growth via nanoparticles9. As expected, more materials with similar morphology have since been synthesized10,11. However, the only metal exhibiting spiral morphology so far remains gold, observed under different experimental conditions5,9,10,12,13,14,15,16,17,18.

Various factors have been considered by researchers to be the cause of gold spiral growth, including the introduction of micelles14 or pre-synthesized substrate immobilized nanoparticles and amines5, bending caused by a net charge repulsion at the nanoplate edge5,16, fungal extract with enzymes present in the cell wall13, yeast extract via the introduction of thiols, amines, phospholipids and organic acids15, “edge adsorption growth” causing chainlike aggregates of thin plates9, or mosaic growth via particle adsorption17. Similar noble metal morphology, namely nanoplates, formed by chemical19,20, electromagnetic5 or electrochemical21 energy, was demonstrated to interact via hydrogen bonding, van der Waals (dipole–dipole) interactions, steric hindrance, ionic interactions, coordination bonds, or other interactions22, forming dimers23, oligomers24, or aggregates9. Particle coalescence25 proceeds also via oriented assembly (OA), observed also for silver exhibiting nanoplate morphology, resulting in a perfect match of the crystal lattice19,20,26,27,28. Theoretical simulations indicated that the coalescence of polyvinylpyrrolidone stabilized nanoplates (for which strong directional forces are absent29) occurs through a side-to-side attachment, which requires less molecular transport to clear the inter-particle gap compared to face-to-face attachment28. Simulation of gold nanoparticles with adsorbed mercaptans indicated that coalescence occurs via expulsion of coated atoms via self-exchange30 and surface diffusion31 from the region of contact without ligand detachment32. In contrast to the well understood OA mechanism, conclusive data on what exactly triggers crystal deformation to induce the spiral morphology of noble metals in aqueous solutions are still elusive.

To elucidate the formation of spiral morphology, we developed a method to synthesize silver in spiral and helicoidal morphologies, which have not been reported thus far. Our method comprises a discrete transformation of silver nanoparticles into silver nanoplates during H2O2 addition33,34, and further into a variety of different morphologies depending on the type and concentration of mercaptan ligand.

Results and discussion

Screening for reagents, concentration, and pH

Mercaptans with different moieties were implemented, and their concentrations were selected to partially cover the surface of spherical, a few nanometer-sized silver nanoparticles (AgNP). The morphologies shown in Fig. 1A were obtained for the same molar ratio of mercaptan, AgNP and H2O2. The addition of mercaptoethanol (ME) or mercaptopropanesulfonate (MPS) resulted in comparably sized silver nanoplates (AgNpt) to preparations without added mercaptan. In comparison, preparations containing added dimercaptoethane (EDT), L-cysteine (CYS), mercaptoacetic acid (MAA), mercaptosuccinic acid (MSA) or 11-mercaptoundecanoic acid (MUA) displayed much larger AgNpt crystals. Differently from other reagents, the addition of 3-mercaptopropionic acid (MPA) resulted in silver nanospirals (AgNsp). Molar ratios between 0.001 and 0.1 MPA/Ag were further investigated by partially or entirely covering the AgNP surface. An initial pH between 3 and 7 was chosen based on MPA functionalized AgNP titration (Supplementary S.1). Distinctly different morphologies shown in Fig. 1B were generated.

Fig. 1: Screening for reagents, concentration, and pH.
figure 1

A SEM images of silver nanoparticles obtained for a molar ratio of mercaptan/Ag = 0.01 and the same amount of 1% H2O2 added, B SEM images of silver nanoparticles obtained after addition of 1% H2O2 for various MPA/Ag and initial pH.

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Expectedly, very low MPA concentrations (MPA/Ag < 0.002) had little effect on AgNpt morphology. At moderate concentrations (0.002 < MPA/Ag < 0.007), larger elongated, irregularly shaped, plate-like particles (silver nanoribbons—AgNrb) were formed, along with a very small fraction of protospirals. Higher MPA concentrations (0.007 < MPA/Ag < 0.025) decreased the number of AgNrb and increased the fraction of AgNsp to yield above 90%, with little effect on particle size (Fig. S2). AgNsp exhibit a conical shape with solid peaks and fragmented larger turns, sometimes approaching trigonal and hexagonal geometry. At high MPA concentrations (MPA/Ag > 0.025), AgNsp with a higher number of turns and increased complexity were formed, as well as silver nanoflowers (AgNfw). Besides, MPA/Ag ratio above 0.09 produced thicker crystals, overlapping spirals, and smaller but more complex AgNfw. Complexity is also affected by initial pH, producing lower complexity at higher initial pH values (Supplementary S.2). Importantly, all tested mercaptans allowed an initial transformation from AgNP into AgNpt, which can be attributed to the properties of silver and H2O2 in aqueous solutions in the absence of substances that form soluble complexes or precipitates35 (Fig. 2H). The redox amphoteric property of H2O2 induced selective silver oxidation and Ag+ redistribution, resulting in AgNpt morphology33, despite the presence of adsorbed mercaptans (Supplementary S.3).

Fig. 2: Adsorption of MPA on AgNP.
figure 2

A Normalized spectra with inset showing change in LSPR(nm) upon continuous addition of 1/1 mM MPA/NaOH to 2 ml AgNP solution; B UV-Vis spectra with inset showing the change in LSPR(nm) upon continuous addition of 1 mM MPA to 2 ml AgNP solution; C change in LSPR and absorbance ratio at 420/380 nm upon MPA addition; D DLS of AgNP with MPA/Ag ratio 0–0.09; E, F optical microscope images of AgNP oligomers and agglomerates. The spot size of the scattered light is much larger compared to the true AgNP size and colors are a consequence of nanoparticle shape and orientation. G Ag(I) thiolate polymer obtained by mixing equimolar amounts of AgNO3 and MPA; H overlayed potential-pH equilibrium diagram for the system Ag-H2O2-HO˙-HOO˙-Ag2S-BH4--H2O at 25 °C and species unity concentration.

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Adsorption of MPA on AgNP

The addition of MPA to AgNP resulted in a localized surface plasmon resonance (LSPR) shift around 400 nm caused by adsorption (insets Fig. 2A, B), affecting the refractive index36. LSPR measurements showed that adsorption started immediately after MPA addition and was completed within approximately 100 s (Supplementary S.4 and Fig. S5). Particle–particle interactions induced dipolar plasmon coupling of the transverse and longitudinal excitations37 as seen from the second peak, indicating the formation of oligomers (Fig. 2B), appearing at MPA surface coverage beyond approximately half of surface saturation (Figs. 2C and S7A). Dynamic light scattering (DLS) showed an initial decrease of the hydrodynamic radius, indicating partial polyvinylpyrrolidone (PVP) displacement by mercaptan (Fig. S8), followed by a substantial radius increase (Fig. S7). Optical microscopy exploiting LSPR (Fig. 2E, F) further supported oligomer formation beyond 0.5 coverage, with a size depending on MPA concentration and colloid aging (Fig. 2D). Although added MPA has been shown to preferentially adsorb on AgNP, once the surface area is saturated, its excess remains in solution, forming an Ag(I) thiolate polymer38. We confirmed this by mixing equimolar amounts of AgNO3 and MPA, resulting in H2O2 stable white gel-like precipitate (Fig. 2G).

Oligomerization under alkali conditions was absent even at high MPA surface coverage (Figs. 2A and S6A, D, E). However, coordination bonds formed by the addition of certain ions, including silver, did lead to the formation of oligomers, even under alkali conditions (Fig. S9).

AgNsp evolution

Evolutionary growth (Fig. 3) was monitored by discrete H2O2 addition, similarly to AgNpt synthesis33. Three different MPA/Ag ratios were investigated, producing three different initial AgNP systems: (low) 0.002 with predominantly single AgNP, (medium) 0.01 combined with oligomeric AgNP, and (high) 0.059, additionally with Ag(I) thiolate polymer.

Fig. 3: Silver nanospiral evolution.
figure 3

Absorbance spectra and analyses for MPA/Ag ratios = 0.002 (A), 0.01 (B), and 0.059 (C) with corresponding SEM images. D Change in pH during growth; E change in extinction and absorbance maximum position during growth; F size comparison between assemblies and AgNpt; G estimated coverage (EC) of AgNpt edge for measured AgNpt with standard deviations next to first assemblies (Supplementary S.7). H Estimated AgNpt transition size for EC = 0.5.

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Previously described morphologies, namely AgNpt, AgNrb, AgNsp and AgNfw, were obtained by increasing the respective initial MPA/Ag ratio. For low MPA/Ag, AgNpt assembly occurred in conditions with significantly raised pH, such as after the addition of 5.4 mL H2O2, as shown by pH profile and UV-Vis analysis (Fig. 3D, E), while before only individual AgNpt were observed (Fig. 3A). The same trend was observed for medium and high MPA/Ag ratios (Fig. 3B, C, E). Interestingly, dendritic morphology also appears, predominantly for the medium MPA/Ag ratio (Fig. 3B). Such morphology is commonly observed during irreversible diffusion-limited aggregation39,40. The observed planar morphology of formed dendrites indicates an OA mechanism (Supplementary S.5 and Fig. S24).

While the MPA/Ag ratio affected the size of AgNsp assemblies (Figs. S2 and S14), the effect of the MPA/Ag ratio was most prominently observed on the assembly/AgNpt size ratio (Fig. 3F) and the size of their “building blocks” (Fig. 3G, H). AgNsp morphology analysis revealed different types of spirals: simple spirals, intergrown, and helicoidal (Fig. 4A–F). Single spirals preserved their handedness on both sides (Fig. 4F, G) while examination of a large set of spirals revealed an approximately equal number of left- and right-handed spirals. XRD revealed a high degree of crystallinity and absence of internal crystal lattice strain (Fig. S15).

Fig. 4: Silver nanospiral formation mechanism.
figure 4

A–E Different morphologies of AgNsp; F images of the same, tilted AgNsp; G optical micrograph of AgNsp from both sides; H–J evolution of AgNsp formation; K–O AgNsp formation mechanism Illustration.

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XPS analysis of AgNsp surface revealed the presence of sulfur, confirming MPA adsorption throughout synthesis (Fig. S16). Raman spectroscopy additionally confirmed PVP displacement and MPA anti conformation after assembly (Fig. S17). The MPA redistribution during evolutionary growth was indirectly tested and confirmed by a seedless experiment (Supplementary S.8).

To elucidate why among the tested mercaptans only MPA produced AgNsp, a closer investigation of MAA, which differs from MPA by a single C atom, was performed. Evolutionary growth with MAA revealed an absence of dendritic morphology (Fig. S22). An important difference between MPA and MAA is in molecule flexibility, which allows adsorbed MPA to occur in two coverage-dependent conformations (Supplementary S.9). Due to the conformation-dependent surface coverage of a single molecule, the switch between the two conformations can be detected via titration (Fig. S21A, B). This was confirmed by titrating 2-mercaptobenzoic acid (2MBA), which can also exhibit two different adsorption conformations41 (Fig. S21B). AgNP with adsorbed 2MBA also produced AgNsp morphology (Fig. S23).

AgNsp formation mechanism

Experimental results suggest the following AgNsp formation mechanism:

  • MPA (or 2MBA) adsorption: Like all mercaptans, MPA (or 2MBA) strongly adsorbs on the AgNP surface, partially displacing PVP and weakening AgNP repulsion (Fig. 4K and Supplementary S.4). At low surface coverages, they interact with silver surface via carboxylate, disabling interparticle interactions.

  • AgNP growth: H2O2 addition causes the transformation from AgNP into AgNpt despite adsorbed MPA (or 2MBA), allowing their further growth (detected by a plasmon shift) (Fig. 4L).

  • Surface area decrease- ligand density increase: AgNpt growth together with a decrease in original AgNP concentration decreases total silver surface area, consequently increasing adsorbed MPA (or 2MBA) coverage, preferentially on reactive edges (Fig. 4L and Supplementary S.7).

  • Ligand conformation switch causing autoaccelerated assembly: Once MPA (or 2MBA) coverage exceeds approximately 0.5 of maximal coverage, the switch in configuration induces the release of a carboxylic group from the silver surface (Fig. S21E). This causes a rise in pH (Fig. 3D) due to the higher pKa value of the carboxylic group and AgNpt assembly (Fig. 4H–J), which stimulate interparticle interactions facilitated by the presence of silver ions (Figs. 4M, N and S9 and S25). The assembly further triggers a sudden decrease of silver surface area and MPA (or 2MBA) redistribution, inducing planar autoaccelerated oriented assembly (AOA), potentially resulting in dendritic morphology due to diffusion limited growth (Supplement S.9).

  • AgNsp formation: Randomly occurring mismatch of dendrite arms extending from a single dendrite represents a spiral nucleation site (Fig. 4I, J, O), further growing via a conventional Burton, Cabrera and Frank mechanism, approaching a morphology defined by its building blocks. Mismatch occurrence is further facilitated by Ag(111) repulsion, indicated by helicoids morphology.

  • AgNsp morphology: The stochastic nature of spiral formation, due to a dendrite arm mismatch, results in a variety of spiral morphologies of equally represented left- and right-handedness, including helicoidal spirals with spaced turns (Fig. 4F).

The proposed mechanism is to some extend similar to earlier suggestions for gold nanospiral formation with respect to attraction between building blocks9,17, but differs from more recent proposals5,16 where spiral formation is attributed to the edge electrostatic repulsion during crystal growth. Contrary to previous studies, however, the occurrence of an intermediate dendrite state formed via AOA, caused by a ligand conformational switch, is believed to be essential for nanospiral formation. As this process occurs in a minute scale caused by a diffusion limiting growth, it is unlikely that a similar mechanism can govern the creation of gold nanospiral morphology, forming over hours or even days. Further in-situ experiments would be required to elucidate the details of the nanospiral formation mechanism on an atomic scale.

Conclusion

Since the threshold coverage of transition is a property of the ligand (MPA or 2MBA in this study), the initial particle size, concentration and amount of added ligand define AgNpt size when AOA occurs (Fig. 3H). AgNsp morphology also appears in the presence of some AgNP synthesis-relevant compounds, demonstrating the robustness of the method (Supplementary S.10). The only precondition for the occurrence of the described mechanism is a strongly adsorbing ligand with coverage-dependent conformation shift, inducing sudden interparticle attraction from the building blocks with substantial difference in face reactivity, directing AOA growth. Once these criteria are met, the proposed approach allows tailoring of the material’s morphology within a few minutes and subsequently its adsorption, catalytic, electric, mechanical, and optical properties1.

Materials and methods

Materials

Polyvinylpyrrolidone (PVP K90), Silver nitrate (AgNO3, 99%), antifoam 204 (A204), ethanol (EtOH, 96%), MSA (99%), sodium 3-mercapto-1-propanesulfonate (MPS, 90%), thioglycolic acid (MAA, 99%), 2-mercaptoethanol (ME, 99%), 4-mercaptophenylacetic acid (MPAA, 97%), 4,4’-dithiodibutyric acid (DTBA, 95%), (Ca(NO3)2·4H2O, 99%), (Ni(NO3)2·6H2O, 97%), (Cr(NO3)3·9H2O 99%), sodium borohydride (NaBH4, 98%) purchased from Sigma-Aldrich. 1,2-ethanedithiol (EDT, 99%), 11-mercaptoundecanoic acid (MUA, 95%), 2-mercaptobenzoic acid (2MBA, 90%) from Tokyo Chemical Industry Co., Ltd. (TCI). 3-mercaptopropionic acid (MPA, 98%), L-cysteine (CYS, 99%) from Carl Roth GmbH. (NaOH, 98%), (HNO3, 65%) from Honeywell Fluka. Hydrogen peroxide (H2O2, 30%) from Carlo Erba reagents. Boric acid (H3BO3, 99.8%) from Fisher Chemicals. All chemicals were used as received without additional purification. Deionized water (DI H2O) was used for all solutions.

Synthesis of AgNP

AgNP colloid synthesis was performed according to a previously published method33. A solution of 440 ml 7 g/l PVP K90, 4.3 ml 1 M AgNO3, and 1 ml of 1 vol% A204/EtOH was prepared in a beaker and then intensively mixed with a Teflon magnetic stirrer. A freshly prepared solution of 25 ml 0.2 M NaBH4 in cold water (4 °C) was added to initiate Ag+ reduction. The colloid was left overnight before dialysis through 3500 molecular weight cut-off tubing (SnakeSkin, ThermoScientific) against DI water until the colloid conductivity dropped below 90% of the initial value. Dialyzed AgNP (dAgNP) were characterized by UV-Vis and stored in a glass bottle wrapped in Al-foil until use.

AgNP colloid properties

LSPR 397 ± 3 nm, extinction coefficient 107–111 AU/(g/l), AgNP diameter based on Mie theory 7.5 ± 4.5 nm. The average AgNP, assuming spherical morphology, has a volume of 221 nm3, surface area of 177 nm2, 12944 Ag0/AgNP, and an estimated specific surface area of 76.3 m2/g. Colloid concentration was 0.97 g/l (8.99 mM Ag0, 4.18 × 1017 AgNP/l), pH was 7 ± 0.4, with 6.6 g/l PVP K90 as steric stabilizer. Estimated mercaptan coverage was calculated based on spherical 7.5 nm AgNP specific surface area (76.3 m2/g) and area for on Ag (111) chemisorbed mercaptan (19.1 Å2/thiolate). Approximately 935 MPA molecules adsorb to 1894 surface Ag atoms, resulting in MPA molecule interaction with approximately 2 surface Ag atoms upon surface saturation. For AgNP used we find that a concentration of MPA 48–834 µM provided an estimated surface coverage of 0.07–1.27.

Silver nanospiral (AgNsp) synthesis

Silver nanospiral (AgNsp) synthesis was performed similarly to the method developed for AgNpt33. Typically, 21 μl freshly prepared 24 mM MPA was added to 5 ml AgNP (0.97 g/l in 6.6 g/l PVPK90). To ensure partial estimated AgNP coverage (14%), MPA was added at 1 mol.% based on the concentration of AgNO3 from which AgNP were synthesized (MPA/Ag = 0.01). The pH of AgNP was adjusted directly after MPA addition with 0.1 M HNO3 or NaOH to pH 3–7. Then 5 ml of freshly prepared 1% H2O2 was added via a submerged capillary with a flow rate of 0.09 ml/min using a syringe pump (AL-1010, World Precision Instruments, USA) while mixing with a Teflon magnet. Instead of dialyzed (AgNP), centrifuged (cAgNpt) were used. Mercaptans with carboxylic acid groups, namely MAA, 3-MPA, MSA, and 11-mercaptoundecanoic acid (MUA) were examined. Additionally, mercaptans with a sulfonic acid group, such as MPS, or with thiol groups only, like 1,2-ethanedithiol (EDT), were examined. A compound with both an amine and carboxylic acid group, namely L-cysteine (CYS), as well as one with a hydroxyl group, namely 2-ME, were also investigated. Molar MPA/Ag ratios ranging from 0.002 to 0.13 (4–270 μl 24 mM MPA for 5 ml AgNP) were investigated, with reaction volumes ranging from 1 to 50 ml and H2O2 concentrations ranging from 1% to 10%. Initial alkali or acidic pH lead in general to smaller and larger particles, respectively. After H2O2 addition, the solutions were stirred for an hour and left undisturbed overnight. Crystals were purified three times with centrifugation (miniSpin, Eppendorf, Germany) for 20 min at 12 kG. Between each centrifugation, crystals were dispersed in DI H2O in an ultrasonic bath (Pro60, Asonic, Slovenia) and used for SEM (see MM Microscopy). For safety, a solution of 10% NaOCl was close when working with mercaptans and used to oxidize waste mercaptans.

Spiral growth evolution

Spiral growth evolution was performed similarly to the method above, with an additional ultrasonic bath filled with 4 l DI H2O used to disperse agglomerates and assist spiral growth. An overhead glass stirring rod was used for mixing. To 50 ml AgNP 0.04, 0.08, 0.21, and 1.24 ml of 24 mM MPA was added for MPA/Ag = 0.002, 0.004, 0.01, and 0.059, respectively. Then 8 ml of 10% H2O2 was added with a syringe pump at a flowrate of 0.09 ml/min. The same experiment was performed with MAA/Ag ratio 0.01 and for MPA/Ag 0.002, 0,01, and 0.091 without ultrasound. Samples with a volume of 1 ml were taken at different H2O2 volumes and immediately purified with centrifugation and deposited on the SEM holder (see MM Microscopy). A similar experiment without ultrasound was performed with 4-mercaptophenylacetic acid (MPAA) and 2-mercaptobenzoic acid (2MBA); however, only one sample was taken after the addition of 4 ml of 10% H2O2.

Seedless AgNsp growth

Seedless AgNsp growth was performed by mixing growth solution made of 3 ml DI H2O, 0.5 ml 28 g/l PVP K90, 0.5 ml 30 mM H3BO3, 0.01 ml 1 M AgNO3, and 0.075 ml 30% H2O2 with the mercaptan 0.1 ml 1 mM MPA (n(SH)/n(Ag) = 0.01) in a 10 ml vial. Growth was initiated by the addition of 75 μl 90 mM NaBH4 or 20 μl of 1 M NaOH with a pipet (Video S1). A top layer where crystals can grow and a bottom layer serving as the reagent pool were formed. Settled crystals were collected after 24 h and purified by centrifugation for microscopy (see MM Microscopy).

Analysis

pH measurement

pH measurement was performed in-situ with a pH micro electrode (InLab Micro, Mettler Toledo) connected to a high-performance liquid chromatography (HPLC) system (ÄKTAexplorer, Uppsala, Sweden).

UV-Vis spectra

UV-Vis spectra were obtained with a spectrophotometer (Tecan, infinite M200Pro, Switzerland) by diluting 5 μl of the sample with 385 μl in a 96-well polystyrene microplate and scanning from 230 to 1000 nm to obtain absorbance (Abs.) UV-Vis spectra from which the wavelength (Wavel.) and extinction of the LSPR peak, namely LSPR(nm), and extinction LSPR(AU) were measured. Because of added reactants, dilutions were considered when drawing and analysing spectra. Data was analysed in Excel using max, min, index, and match functions. Normalization based on LSPR(AU) (Absorbance/LSPR(AU)) was used to better visualize LSPR shifts.

UV-Vis absorbance

UV-Vis absorbance was measured discretely on HPLC system at 380, 420, and 490 nm. This was done by withdrawing the colloid at 0.2 ml/min through the UV-Vis detector of the HPLC, enabling measurement of absorbance at three wavelengths. The absorbance ratios (abs. ratio) for absorbance at 420 and 380 nm (420/320) was calculated to monitor absorption extent as it closely matched the change in LSPR(nm) from UV-Vis spectra. The abs. Ratio at 490 and 380 nm (490/380) was used for oligomerization onset.

MPA functionalized dAgNP titration

MPA functionalized dAgNP titration was performed on HPLC with 5 ml dAgNP by first adding 0.75 ml of a solution of 6 mM MPA in 18 mM NaOH to obtain pH ~10 for MPA/Ag = 0.09 (1.4-times the estimated surface coverage). Three millimolar HNO3 was continuously added with a syringe pump at a flow rate of 0.09 ml/min while continuously measuring pH until a pH of ~3.5 was reached (see MM pH measurement).

Monitoring of MPA adsorption on dAgNP

Monitoring of MPA adsorption on dAgNP was performed on HPLC to measure pH (see MM pH measurement) and absorbance (see MM UV-Vis absorbance) with 30 ml of 15x diluted dAgNP to which different amount of MPA was added (27, 54, 81, 108, 135 µl of 24 mM MPA), corresponding to 0.5, 1, 1.5, 2, and 2.5 times the estimated coverage (Supplementary S.7). Dead time for UV-Vis was measured to be approximately 1 min (coinciding with the change in pH) and was subtracted from UV-Vis to match the in-situ pH response.

Reaction of dAgNP with mercaptan

Reaction of dAgNP with mercaptan was performed on HPLC to measure pH (see MM pH measurement) with 2 ml dAgNP by the addition of 3 ml 1 mM mercaptan dissolved in 1 mM NaOH via a submerged capillary connected to a syringe pump with a flowrate of 0.09 ml/min. Consecutive addition of 3 ml 1 mM HNO3 and 6 ml 1 mM NaOH at the same flowrate was used to change the pH after adsorption. UV-Vis was measured separately for every 0.2 ml of added reagent (see MM UV-Vis spectra). A similar experiment measuring only pH was also performed with either (A) 3 ml of 1 mM MPA in 1–2 mM NaOH added to 2 ml dAgNP, (B) 3 ml of 1 mM MAA, MPA, MPAA, 2MBA in 1.8 mM NaOH added to 2 ml dAgNP, and (C) 3 ml of 0.5 mM DTBA, 1 mM MUA, MAA in 1.8 mM NaOH and 1 mM ME, EDT in 0.8 mM NaOH added to 2 ml dAgNP. Another similar experiment was performed with AgNpt for MPA/Ag ~0.13 with no NaOH addition. SEM images were taken before and after MPA addition to centrifuged AgNpt with MPA/Ag ~0.33 immediately deposited onto SEM holder without additional purification were also conducted (see MM Microscopy).

Dynamic light scattering (DLS)

Measurements were performed to determine the size of AgNP functionalized with MPA using a particle analyser (Anton Paar, Litesizer 500, Graz, Austria). First, the optimal concentration of AgNP was determined by measuring DLS for different AgNP dilutions. One sample was measured 10 times for 1 min and the average value of 6 measurements was calculated. The appropriate amount of 24 mM MPA or ME or NaMPA (n(NaOH)/n(MPA) = 1) was added to 1.5 ml of AgNP colloid and then diluted before measurement. The effect of salts was tested by adding the appropriate amount of 0.1 M solutions of Ag(I), Ca(II), Ni(II), and Cr(III) nitrate to AgNP to a final concentration of 1 mM for NaMPA/Ag = 0.01.

Adsorption/desorption of PVP and MPA on AgNP

Adsorption/desorption of PVP and MPA on AgNP was studied on HPLC with a porous high internal phase polymer (polyHIPE) support prepared and characterized33 with quaternary amine (QA) ligand42. Briefly, AgNpt were immobilized on the porous monolithic matrix to allow flow-through operation. Injections of 0.5 ml 10 mM NaBH4 in 1 mM NaOH at a rate of 1 ml/min were used to regenerate AgNpt. Then 0.5 ml of 1 g/ml PVP K90 was injected until saturation, followed by 0.5 ml injections of 20 mM MPA. This cycle was also repeated without the PVP step. An increased flowrate of 2 ml/min had no effect on the measurements, suggesting fast MPA binding.

Ionic charge shading by Ag+

Ionic charge shading by Ag+ was tested by adding 28 µl 24 mM MPA in 24 mM NaOH (NaMPA) to 1 ml AgNP to achieve an estimated MPA coverage of 1.04. Spectra for AgNP were recorded after reagent addition and after AgNO3 addition to a final concentration of 1 mM (see MM UV-Vis spectra). With increasing NaOH equivalents at constant AgNO3 (1 mM), second peak formation became less pronounced, suggesting ion loss due to Ag2O formation (data not shown).

NaMPA immobilization

NaMPA immobilization was performed on HPLC with a 15x diluted AgNP solution. The appropriate amount of 1 mM MPA in 1 mM NaOH was added to achieve an estimated coverage saturation of 1.04. The colloid was then pumped at 3 ml/min through a high internal phase emulsion polymer matrix (polyHIPE) with QA functionality. After washing with DI water, loading was increased by first pumping 10 ml of 1 mM AgNO3, followed by a 10 ml DI water wash before colloid loading.

X-ray photoelectron spectroscopy (XPS) measurements and depth profiling

X-ray photoelectron spectroscopy (XPS) measurements and depth profiling were conducted using a Versa Probe 3 AD (PHI, Chanhassen, USA) equipped with a monochromatic Al Kα X-ray source. The source operated at an accelerating voltage of 15 kV and an emission current of 13.3 mA. Powder samples were mounted on double-sided Scotch tape and positioned at the center of the XPS holder. Spectra were acquired for each sample over a 1 × 1 mm analysis area, with the charge neutralizer activated during data collection. Survey spectra were measured using a pass energy of 224 eV and a step size of 0.8 eV. High-resolution (HR) spectra were recorded with a pass energy of 27 eV and a step size of 0.1 eV. To ensure high-quality spectral data with a good signal-to-noise ratio, at least 10 sweeps were performed for each measurement. Argon ion (Ar+) sputtering was used for surface cleaning and depth profiling. The ion beam operated at an acceleration voltage of 2 kV over a 2 × 2 mm sputtering area with an estimated sputtering rate 4.5 nm/min. Spectral analysis was carried out using MultiPak 9.9.1 software, and Shirley background correction was applied to all spectra.

X-ray diffraction (XRD)

AgNsp samples for X-ray powder diffraction (XRD) were prepared as described in Spiral growth evolution for MPA/Ag = 0, 0.005, 0.01, and 0.02. AgNsp were purified with centrifugation at 1000 g for 2 min for 5 times and redispersed in 1 mM NaOH to remove most aggregates and finally redispersed in DI water. Grinded QA modified polyHIPE prepared according to the previous report42, was then added to the colloids. This was performed to randomize the crystals orientation. The mixture was well dispersed with vortex mixing and ultrasound and dried in an oven at 65 °C. XRD was measured on dried samples, with Cu-Kα radiation (λ = 1.54 Å) in the range 2θ between 10° and 80° using an PANalytical X’Pert X-ray diffractometer. A qualitative phase analysis of the samples was done using the Crystallographica Search-Match (Oxford Cryosystems, UK) software program.

Raman spectroscopy

Raman spectra were collected using a Bruker Bravo™ Raman spectrometer, equipped with DuoLaser™ technology (785 and 852 nm) and Sequentially Shifted Excitation (SSE™) for fluorescence suppression. Samples were measured directly without preparation. The instrument operated with automatic settings for integration time and laser power. For each sample, three spectra were acquired and processed using Bruker OPUS software, with baseline correction and smoothing applied where necessary.

Microscopy

Microscopy was performed with a field-emission scanning electron microscopy (FE-SEM, Zeiss ULTRA plus, ZEISS, Oberkochen, Germany). Aluminum carriers were polished using Nigrin car polish and a cotton cloth. The carriers were then sonicated in 20% EtOH three times and dried in a stream of air. Centrifuged sample volumes of 0.5 µl were then deposited around the edge of the preheated carrier and completely dried at 60 °C overnight. 10–15 kV accelerating voltage and a working distance of approximately 5 mm were used for all samples. Images were analysed with ImageJ software. For optical microscopy, Zeiss LSM 700 (Oberkochen, Germany) was used. Centrifuged sample volumes of 1 µl were deposited on glass slides and observed using bright and darkfield with reflected and transmitted light. Images were analysed with ImageJ software.