Molecular-level control over plasmonic properties in silver nanoparticle/self-assembling peptide hybrids

Abstract

The plasmonic properties of AgNP arrays are directly controlled by AgNP size, shape, and spatial arrangement. Reported here is a strategy to prepare chiral AgNP arrays templated by two constitutionally isomeric aromatic peptide amphiphiles (APAs), K_SC’EK_S and C’EK_SK_S (K_S= S-aroylthiooxime-modified lysine, C’= citrulline, and E=glutamic acid). In phosphate buffer, both APAs initially self-assembled into nanoribbons with a similar geometry. However, in the presence of silver ions and poly(sodium 4-styrenesulfonate) (PSSS), one of nanoribbons (K_SC’EK_S) turned into nanohelices with a regular twisting pitch, while the other (C’EK_SK_S) remained as nanoribbons. Both were used as templates for synthesis of arrays of ~8 nm AgNPs to understand how small changes in molecular structure affect the plasmonic properties of these chiral AgNP/APA hybrids. Both hybrids showed improved colloidal stability compared to pure AgNPs, and both showed enhanced sensitivity as surface-enhanced Raman spectroscopy (SERS) substrates for model analytes, with nanohelices showing better SERS performance compared to their nanoribbon-counterparts and pure AgNPs.

Silver nanoparticles (AgNPs) are suitable substrates for catalysis,^1–2 sensing,^3–4 and antimicrobial applications,^5–6 and their properties depend heavily on their size, shape, and spatial arrangement.^7–8 Therefore, synthesis of AgNPs with well-defined dimensions and controlled spatial arrangements allows researchers to tune the properties of these materials for specific functions. Toward this end, materials including carbon nanotubes,² polymers,^9–11 DNA,^12–13 and peptides^14–15 have been used as scaffolds to direct the growth or assembly of AgNPs, relying on strong interactions between AgNPs or their precursors and functional groups on these scaffolds. Among these, self-assembled peptides are particularly attractive platforms for AgNP growth^16–19 because of their chemical tunability and their capacity to self-assemble into a wide variety of morphologies, including nanoribbons, nanosheets, and nanohelices, among others.^20–24 For instance, nanofibers assembled from aldehyde-functionalized peptide amphiphiles were used to template the growth of AgNPs in the presence of Tollens’ solution.¹⁶ Particularly intriguing are self-assembled peptide-based helical nanoribbons,¹⁷ which organize achiral AgNPs into chiral assemblies. However, the structure-property relationships among the molecular composition of the template, the spatial arrangements of the AgNPs, and the resultant plasmonic properties of the hybrids remain elusive.

We recently discovered a class of constitutionally isomeric aromatic peptide amphiphiles (APAs) that self-assemble into different morphologies depending on the specific peptide sequence.²⁵ The key component of these APAs, each made of four amino acids, is the inclusion of two lysine residues that contain aromatic S-aroylthiooxime (SATO) functional groups²⁶ attached to their ε-amines.^27–31 We viewed these APAs as templates for AgNP growth, which would enable the AgNP/APA hybrids to retain the chiral imprint from the APA nanostructures. Because these APAs are constitutional isomers, we envisioned that we could control the spatial arrangements of AgNPs and therefore, the plasmonic properties of the hybrids by simply changing the order of amino acids. In this context, we report here that silver salts induce a morphological transition in two constitutionally isomeric APAs, which we used as the templates for AgNP growth. Our results showed that the APA morphology affected the spatial arrangements of AgNPs, which influenced the plasmonic properties of the hybrids, and as a result, their sensitivity as substrates for surface-enhanced Raman spectroscopy (SERS).

We synthesized two constitutionally isomeric APAs that each contained one glutamic acid (E), one citrulline (C’), and two SATO-modified lysine (K_S) residues: K_SC’EK_S and C’EK_SK_S (Figures 1A–B, S1–2). Based on our previous studies on APAs with similar structures,²⁵ we expected that K_SC’EK_S and C’EK_SK_S would form different self-assembled morphologies due to the different arrangements of amino acids in their structures. However, we were surprised to find that both APAs assembled into one-dimensional nanostructures with similar morphologies based on transmission electron microscopy (TEM) observations (Figures 1C and 1D). K_SC’EK_S formed nanoribbons with average widths of 7 ± 1 nm and lengths of a few micrometers (Figure 1C). C’EK_SK_S also assembled into micrometer-long nanoribbons with average widths of 6 ± 1 nm, (Figure 1D). Next, we added silver nitrate (AgNO₃, 0.5 mM) and poly(sodium 4-styrenesulfonate) (PSSS, 0.5 mg mL⁻¹) to induce AgNP formation, which we typically use in AgNP synthesis.³² Interestingly, upon addition of AgNO₃ and PSSS, the K_SC’EK_S nanoribbons changed into micrometer-long nanohelices with an average diameter of 6 ± 1 nm and a regular twisting pitch of 29 ± 5 nm (Figure 1E). In sharp contrast, APA C’EK_SK_S retained its nanoribbon morphology after adding AgNO₃ and PSSS, although the average length decreased (Figure 1F). The distinct morphology differences between K_SC’EK_S and C’EK_SK_S before and after salt addition exemplify how small changes in APA sequence can lead to dramatically different self-assembly behaviors.

Figure 1.

(A and B) Schematic illustrations of the chemical structures of the two isomeric APAs. (C–F) TEM images of (C) nanoribbons formed by K_SC’EK_S and (D) C’EK_SK_S in 10 mM PB; (E) nanohelices derived from K_SC’EK_S and (F) short nanoribbons assembled by C’EK_SK_S in 10 mM PB containing AgNO₃ and PSSS.

Next, molecular packing within self-assembled APAs before and after addition of AgNO₃ and PSSS was assessed by circular dichroism (CD) spectroscopy. Before adding salts, both K_SC’EK_S and C’EK_SK_S displayed a classical β-sheet secondary structure, along with a strong SATO absorption (Figure S3). Interestingly, the CD spectra were nearly identical mirror images; this inversion likely resulted from different handedness of the assemblies.^{27, 33} When AgNO₃ and PSSS were added to the solution, the spectrum changed into a random coil-like structure for K_SC’EK_S (the same as when only PSSS was added to the solution). However, the spectrum did not change significantly for C’EK_SK_S. These changes were consistent with TEM observations, where K_SC’EK_S showed significant morphological changes after salt addition, but C’EK_SK_S did not.

We next used these two nanostructured templates to synthesize AgNP/APA hybrids in a two-step process (Figure S4A). In the first step, we added AgNO₃ and PSSS to a K_SC’EK_S solution, followed by the reducing agent NaBH₄. The solution changed from colorless to yellow, suggesting the formation of small Ag seeds (AgSDs).³² A UV-Vis spectrum showed a peak near 405 nm (Figure S4B), consistent with the characteristic plasmonic response for non-aggregated, spherical AgNPs smaller than 10 nm.³⁴ In the second step, additional AgNO₃ solution was added dropwise into an aliquot of AgSDs (50–1000 μL) and ascorbic acid. During this second growth step, the color of the solution became bright yellow, with the peak absorption remaining at 405 nm (Figure S4B). We determined that both a minimum amount of AgSDs and PSSS were required for maintaining the K_SC’EK_S nanohelix morphology in the resultant AgNP/K_SC’EK_S hybrids. Addition of too little AgSD solution resulted in the formation of nanoribbons (Figure S5), as did excluding PSSS from the synthesis (Figure S7). We then used this optimized procedure to prepare AgSDs and AgNPs templated by the C’EK_SK_S nanoribbons, which showed the same color change in each step as the AgSDs and AgNPs templated by the K_SC’EK_S nanohelices.

TEM was then used to examine the morphologies of the AgNP/APA hybrids and measure the sizes of the AgSDs and AgNPs in both samples. The growth of AgSDs and AgNPs had little influence on the original morphologies of these two different self-assembled APAs (Figures 2A–D). In the presence of K_SC’EK_S nanohelices, the diameter of the AgSDs was 2.8 ± 0.5 nm (Figure 2A). After the second round of Ag growth, the diameter of the AgNPs increased to 7.6 ± 0.9 nm (Figure 2C). When C’EK_SK_S nanoribbons were used as the template under the same growth conditions, AgSDs and AgNPs formed with diameters of 2.9 ± 0.6 nm (Figure 2B) and 8 ± 1 nm (Figure 2D), respectively. Although the sizes of the AgSDs and the AgNPs were similar on both the nanohelices and the nanoribbons, the AgSDs and AgNPs were distributed mostly within the K_SC’EK_S nanohelices (Figures 2A and 2C), while those templated by the C’EK_SK_S nanoribbons distributed at the edges of the nanoribbons (Figures 2B and 2D). We also prepared AgNP/K_SC’EK_S hybrids without PSSS, which as we noted above was vital for nanohelix formation (Figure S7). These AgSD and AgNP arrays had similar appearance and dimensions (2.6 ± 0.9 nm for AgSDs and 8 ± 1 nm for AgNPs) to the nanoribbon-forming AgNP/C’EK_SK_S hybrids. These results demonstrate that both APAs functioned as templates for AgNP growth, regulating the spatial arrangement of these nanoparticles.

Figure 2.

(A-D) TEM images of (A) AgSD/K_SC’EK_S nanohelices, (B) AgSD/C’EK_SK_S nanoribbons, (C) AgNP/K_SC’EK_S nanohelices, and (D) AgNP/C’EK_SK_S nanoribbons. Inserts in the top right corners of panels A–D show zoomed-in images of the areas outlined by the dashed red rectangles. (E and F) CD spectra of (E) AgNP/K_SC’EK_S nanohelices and (F) AgNP/C’EK_SK_S nanoribbons.

CD spectroscopy allowed us to investigate the chirality of the resultant hybrids. The CD spectrum of AgNP/K_SC’EK_S displayed three negative peaks at 386, 407, and 471 nm (Figure 2E), whereas that of AgNP/C’EK_SK_S exhibited only a broad negative peak with a minimum at 414 nm (Figure 2F). For an achiral assembly of AgNPs, a flat CD spectrum would be expected, so these spectra confirm that chirality was transferred from the APA nanoassemblies to these hybrid structures, similar to AgNPs templated by DNA.¹³ In addition, the absorption beyond 470 nm was much stronger for AgNP/K_SC’EK_S than for AgNP/C’EK_SK_S; this spectral feature may indicate a stronger interaction between AgNPs and K_SC’EK_S nanohelices than C’EK_SK_S nanoribbons.³⁵

Next, we evaluated the stability of the AgNP/APA hybrids through multiple lyophilization cycles, as this property is critical for storage, transportation, and further practical applications. The stability was assessed by measuring the intensity of the localized surface plasmonic resonance (LSPR) absorption of the AgNPs at 405 nm, which reflects the concentration of dispersed AgNPs in solution. The absorbance of all hybrids and pure AgNPs of similar size decreased with each lyophilization cycle (Figures 3A and S8). Specifically, the absorbance of the helical AgNP/K_SC’EK_S hybrids decreased to 80% of its original value after one lyophilization cycle, while nanoribbon-forming AgNP/C’EK_SK_S hybrids decreased to 55%. Similarly, the absorption of AgNP/K_SC’EK_S nanoribbons prepared without PSSS decreased to 58% after one lyophilization cycle. The absorption of pure AgNPs (without any APA template) after one cycle decreased to 5% of its original value. Notably, all the hybrids could be re-hydrated and re-suspended after several cycles of lyophilization, while pure AgNPs could not. We conducted similar colloidal stability studies by adding increasing amounts of salt (PB) to these three types of AgNP/APA hybrids along with pure AgNPs (Figure S9). Results showed that all AgNP/APA hybrids tolerated salt better than pure AgNPs.

Figure 3.

(A) Colloidal stability of all groups studied in the present work against lyophilization. Comparing the decay rates of the intensities of the LSPR absorbance peak corresponding to AgNPs (λ_LSPR ≈ 405 nm) indicates the resistance of AgNPs to aggregation. The peak intensity prior to lyophilization serves as a reference for normalization (100 % relative absorbance). Data were fitted with an exponential model (See Supporting Information for details). (B) SERS spectra of 10⁻⁴ M RhB collected on AgNP/APA hybrids with different morphologies, pure AgNPs, and pure APAs. Spectra of controlled samples were intensified as indicated for better visualization.

Finally, given the different spatial arrangements of AgNPs on the self-assembled APAs, we asked whether these hybrids could be used as substrates for SERS. Specifically, we investigated whether APA morphology in these AgNP/APA hybrids would affect SERS sensitivity. We selected rhodamine B (RhB) and 2,2’-bipyridine (Bpy) as model SERS analytes. Notably, for both analytes, the helical AgNP/K_SC’EK_S hybrids outperformed all other materials investigated. In the case of RhB, it enhanced the characteristic Raman bands to a much greater extent than the two nanoribbon-forming controls, AgNP/C’EK_SK_S and AgNP/K_SC’EK_S without PSSS (Figure 3B). Additional control systems including pure AgNPs (no APA template) as well as the APAs K_SC’EK_S and C’EK_SK_S without AgNPs were also investigated for comparison. The pure AgNPs displayed a weak SERS signal, while the APAs alone showed no discernable SERS signal. We also observed the same trend of SERS enhancement when Bpy was used as the analyte (Figure S10).

These results indicate that the spatial arrangement of AgNPs into the helical configuration provided by K_SC’EK_S is key for superior SERS enhancement over its nanoribbon-forming counterparts because the size and shape of the AgNPs investigated were the same. We hypothesize that AgNPs within the grooves of K_SC’EK_S nanohelices were better immobilized and provided a more favorable 3D arrangement for SERS than the AgNPs on the nanoribbons (C’EK_SK_S or K_SC’EK_S without PSSS). Compared to commonly used SERS substrates that are either plasmonic NPs grafted with ligands or lithographically patterned metal surfaces,^36–39 our constructs provide unique particle arrangements, require negligible energy input, possess high colloidal stability, and are dispersible in biological media.

In summary, we have reported a simple strategy to construct chiral AgNP hybrids from two APAs, K_SC’EK_S and C’EK_SK_S, both of which initially assembled into nanoribbons with similar dimensions. However, when silver ions and PSSS were added, one of nanoribbons (K_SC’EK_S) transformed into nanohelices. Both nanostructures served as effective templates for AgNP growth, and the resulting hybrids showed improved stability over pure AgNPs. We found that these hybrid materials were suitable as SERS substrates, with the nanohelix-forming hybrids exhibiting a superior signal enhancement driven by the unique spatial arrangement of AgNPs compared to their nanoribbon counterparts. This work highlights how small changes in molecular structure can dramatically alter plasmonic properties. We believe this strategy reveals novel ways to construct sophisticated peptide-based nanostructures for biomedical applications such as biomarker detection.