Green synthesis and fabrication of an electrochemical and colorimetric sensor based on self-assembled peptide-Au nanofibril architecture

1 Introduction

Due to the unique optical, thermal and biophysical properties, gold nanoparticles (AuNPs) have been widely studied for various applications in recent decades (Daniel and Astruc, 2004). AuNPs in diameters of around 10–50 nm are usually chemically reduced from Au³⁺ in aqueous solution (Daniel and Astruc, 2004; Saha et al., 2012). As the non-capping AuNPs are intending to aggregate in aqueous solution and then lose their chemical and physical properties, many efforts have been performed to stabilize the chemically reduced AuNPs by encapsuling AuNPs using porous inorganic materials (Guo and Suslick, 2012; Song et al., 2015; Yu et al., 2017a), graphene/graphene oxide (Li et al., 2016, 2017; Myung et al., 2011; Thirumalraj et al., 2017) and polymers (Peng et al., 2012; Rahme et al., 2013). As potential surface ligands, some biomolecules, such as DNA (Jin et al. 2003), proteins (Wei et al., 2010; Xie et al., 2009), and peptides (Hamley et al., 2014; Wang et al., 2016; Wu and Jin, 2010) have also been utilized for stabilizing AuNPs due to their high biocompatibility, easy modification, and excellent self-assembly ability to form hierarchical nanostructures, which show fascinating optical, electrical, and biological properties (Li et al., 2013; Zeng et al., 2016).

To induce the assembly of AuNPs to a supramolecular nanostructure, a few typical strategies have been utilized. For example, a self-assembled chiral nanofibrils have been employed as building blocks for organizing one-dimensional AuNPs structure (Jung et al., 2014). Layer-by-layer self-assembly inorganic nanotubes have also been used as templates for synthesis the monodispersed AuNPs (Xiao, 2012). In another case, Ding et al. synthesized Au–ZnO heteronanostructures by dielectrophoretic assembly (Ding et al., 2015). These above introduced methods induced the further organization of synthesized AuNPs on a given template, and requiring additional reducers. Due to biomineralization and self-assembly abilities, amyloid peptide has become a promising strategy of creating assembled architecture of AuNPs (Chen and Rosi, 2010). Firstly, peptide can induce biomineralization of various inorganic nanomaterials, including hydroxyapatite (Li et al., 2014; Wang et al., 2015), TiO₂ (Sewell and Wright, 2006), CaCO₃ (Volkmer et al., 2004), CdS (Peelle et al., 2005), Ag (Wang et al., 2014), and Au NPs (Nystrom et al., 2016; Wang et al., 2012; Zhang et al., 2017). Secondly, amyloid peptides can lead nanoparticles self-assemble into nanofibrils. As peptide nanofiber (PNF)-based materials have been designed and investigated in recent decades (Assenza et al., 2014; Bolisetty and Mezzenga, 2016; Li et al., 2011; Wei et al., 2017) sequence designed amyloid peptide has become an emerging approach of fabricating multifunctional supramolecular structures. However, the synthesis and assembly of AuNPs usually need complicated procedures, and require additional chemicals. Therefore, the development of green and efficient methods for the synthesis of hierarchical architecture of AuNPs nanofibers is of great interest. We report a green and efficient method for building supramolecular nanofibers formed by peptide and AuNPs.

In this work, we demonstrated a green synthesis and fabrication strategy for building self-assembled PNF-AuNPs nanofibril sensor. AuNPs are usually synthesized by harsh chemical reduction and then stabilized by capping agents (Daniel and Astruc, 2004) The peptide we designed plays three roles: reduction agent, stabilizer and self-assembly template. The synthesis and fabrication procedures are simple and calm, without complicated post-treatment. The multifunctional motifs of peptide include three special amino acid sequences. One is biomineralization motif (CCY), the other one is a structural motif (AEAKAEAKAEAKAEAK), and a cellular recognition motif (RGD). The created PNF-AuNPs architectures exhibit excellent electrochemical and colorimetric sensing performances for detecting H₂O₂ and Hg²⁺, respectively.

2 Results and discussion

Scheme 1 presents the proposed synthesis mechanism of PNF-AuNPs. After mixing AuCl₄⁻ ions with peptide and adjusting the pH value of solution to 12, the functional amino acid sequences, CCY, offers the side group of tyrosine to reduce AuCl₄⁻ ions to Au nanoclusters, and then the formed Au nanoclusters are captured and protected by the thiol side group of cysteine, to form peptide-protected AuNPs. Therefore, AuNPs can be synthesized and organized by adjusting the concentration of peptide monomers.

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Scheme 1 Schematic of synthesis and self-assembly procedure of PNF-AuNPs. The green arrows of PNF-AuNP refer to the β-sheet structure of self-assembled PNF-AuNPs.

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It has been reported that the size of AuNPs is notably associated with pH value and the molar ratio of Au³⁺ ions to thiol groups (Cui et al., 2011). In fact, as the thiol group enrich on the surface of self-assembled PNFs, the local thiol concentration may be difficult to be controlled, which could lead to a low yield and even aggregation of AuNPs. Therefore, in this work, we employed a one-pot synthesis strategy, in which peptide-AuNP monomers were synthesized firstly, and then the peptide-AuNPs self-assembled under the driving forces of peptide self-assembling. Based on the morphology of self-assembled PNFs (Fig. 1a) and our previous experience (Su et al. 2015; Zhang et al., 2017). The Au nanoclusters may nucleate on peptide monomers at first, and then those Au nanoclusters are captured by -SH side groups, further growing and forming AuNPs protected by peptide ligands. We suggest that the peptide-AuNP monomers self-assembled into a fibrous structure through the hydrophobic interaction among the assembly-specific domain of the peptide sequence.

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Fig. 1 TEM images of (a) self-assembled PNFs, (b) peptide-AuNPs monomers, and (c and d) PNF-AuNPs. Figure e and f show the diameter distributions of (e) peptide-AuNPs and (f) PNF-AuNPs. The average diameters are 9.55 ± 0.6 nm and 15.55 ± 0.8 nm, respectively. We measured at least 150 samples for each type of nanoparticle. PNFs sample was diluted to 0.5 mg/mL. Peptide-AuNPs and PNF-AuNPs sample was diluted to 1 mM (HAuCl₄).

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Transmission electron microscopy (TEM) was utilized to characterize the morphologies of both peptide-AuNPs (Fig. 1b) and PNF-AuNPs after their self-assembly (Figs. 1c, d, and S1). The self-assembly process is driven by the hydrophobic force and hydrogen bonds between the alternative arrays of hydrophobic and hydrophilic amino acids, which leads to the formation of β-sheet secondary structure (Bowerman and Nilsson, 2012; Mayans et al., 2015), inducing the assembly of AuNPs. To confirm the type of secondary structure, FT-IR measurements was performed (Fig. S2). Amide I peak was found in 1630–1690 cm⁻¹, indicate exist of β-sheet structure (Rangnekar et al., 2007; Zhou et al., 2009). The fractions of several types of secondary structures were further revealed by circular dichroism (CD) spectra (Fig. S3 and Table S1). Antiparallel β-sheets formed the PNF-AuNPs mainly. The AuNPs grow and distributed along the long axis of formed PNFs. The size of AuNPs slightly is increased after the assembly (from 9.55 ± 0.6 nm to 15.55 ± 0.8 nm) (Fig. 1e and f), proving that the self-assembly of peptide chains induce the growth and merge of different Au nanoclusters. Notably, there are several free AuNPs can be observed in TEM images, we assume they are the shorter nanofibers and some free peptide-AuNPs which not assembled with others. Dynamic Light scattering test was performed to measure the fractions of that part (Fig. S4 and Table S2).

The self-assembly of AuNPs is related to not only the β-sheet formation, but also the formation of disulphide bonds, as identified by X-ray photoelectron spectroscopy (XPS) measurement. Fig. 2 demonstrates the XPS results of peptide-AuNPs (Fig. 2a –c) and assembled PNF-AuNPs (Fig. 2d –f). Compared with peptide-AuNPs monomers, the self-assembled PNF-AuNPs exhibit a change on the signal of S2p, which suggest that there are some –SH groups forming disulfide bonds after the self-assemble process, indicating the formation of disulfide bonds after the self-assemble process. Those disulfide bonds may lead to a more steady chemical and mechanical structure of formed nanofibril architectures (Khadka and Haynie 2012).

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Fig. 2 XPS results of peptide-AuNPs monomers (a–c), and PNF-AuNPs (d–f). (b, e) are the results of Au4f, and (c, f) are the results of S2p, respectively.

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There is a great interest for building hierarchical self-assembled structures of noble metal NPs due to their excellent sensitivity towards intermediate metabolite, such as hydrogen peroxide (H₂O₂) and fatal heavy metal ions (Gao et al., 2015, 2013). It can be predicted that the self-assembled PNF-AuNPs would display a remarkable enhancement on the stability and dispersity of AuNPs, leading to the excellent sensing performances. Herein, dual sensing applications based on electrochemical sensor and colorimetric assay were presented.

As an important intermediate production in biosystem, H₂O₂ sensing are attracting continuous concentration in recent decades (Yu et al., 2017b; Zhang et al., 2013, 2015), especially as nanoprobes based on biological-friendly materials for in vitro and intracellular assays. In this work, PNF-AuNPs was first employed to modify the glassy carbon electrode (GCE) and fabricate an electrochemical biosensor (Fig. 3a). The performance of sensing H₂O₂ was measured by three-electrode system with modified GCE as the work electrode, platinum wire electrode as the counter electrode, and saturated calomel electrode as the reference electrode. These experiments were performed under room temperature.

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Fig. 3 Electrochemical sensing properties of PNF-AuNPs. (a) Schematic of PNF-AuNPs-based electrochemical sensing towards peroxide. (b) CV measurements towards 5 mM H₂O₂, black and red lines refer to PNF-modified electrodes and PNF-AuNPs modified electrodes, respectively. (c) Current response of PNF-AuNPs towards continuingly addition of H₂O₂ solution. (d) Linear-fit results of Current-Concentration relationship, R² = 0.997. (e) Selectivity property of PNF-AuNPs. Arrows refers to one addition of 5 mM H₂O₂, 1 mM glucose, 1 mM DA, 0.1 mM UA, 1 mM AA, and 5 mM H₂O₂, respectively. Fig. (c, e–g) were performed under the voltage of −0.40 V, scan rate 0.1 V/s.

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Fig. 3b displays the CV curve of PNF-AuNPs modified GCE in 5 mM H₂O₂ solution. Compared to PNF-modified electrode, a rising reduction peak at −0.36 V shows, indicate that PNF-AuNPs modified sensitivity to H₂O₂. Fig. 3c shows the amperometric test with manually adding H₂O₂ and increasing the concentration of H₂O₂ from 0.05 to 27.85 mM, under the scan potential of −0.36 V. With the increasing of H₂O₂ concentrations from 0.1 to 25.85 mM, the curves show fast, linear current response towards the increasing H₂O₂ concentration, as shown in Fig. 3d. The fitting of the concentration-amperometric relationship revealed a linear response with R² = 0.997, and the detection limit was calculated to be 3.43 μM (S/N = 3). The selectivity and anti-interference ability were further measured in same conditions by c (Fig. 3e). The curve shows slightly change when DA was added, besides there is no any obvious current change towards other chemicals. After adding the interference chemicals, 5 mM H₂O₂ was added, again, the sensor shows intense current response towards H₂O₂. Following tests indicated that the fabricated PNF-AuNPs based sensor show very good reused ability and stability (Fig. 3f and g). Within 7 times reuse in one day and repeat use in 2 week, the changes of current response were within 3 × 10⁻⁶ A, which shows the good stability of the fabricated sensor.

Colorimetric detection of heavy metal ions like Hg²⁺ (Duan et al., 2014), Cd²⁺ (Zhang et al., 2012), and Cu²⁺(Xu et al., 2010), has been an attractive issue in healthcare and environmental monitoring. Compared to other sensing techniques, colorimetric assay is visible for naked eye and convenient to operate. It is reported that cysteine-capped AuNPs possess high selectivity towards Hg²⁺ (Du et al., 2013), due to the strong interaction between Hg²⁺ ions and cysteine. When Hg²⁺ ions were added into well-dispersed AuNPs solution, the protective group (—SH) would coordinate with Hg²⁺ and form a cross-linked network, while AuNPs that lost the protection of thiol would aggregate in water solution, causing the colorimetric change (Scheme 2). As a result, the red solution would darken and turn into blue or purple. The concentration-color relation can be observed by naked eye, and linear relation between concentration and color changing can be measured by UV–Vis spectroscopy. Similar with independent dispersed AuNPs solution, the self-assembled PNF-AuNPs shows the colorimetric response towards heavy metal ions due to the anti-aggregation mechanism (Duan et al., 2014). Thus we tested the selectivity of PNF-AuNPs solution towards metal ions.

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Scheme 2 Schematic of self-assembled PNF-AuNPs based colorimetric assay towards Hg²⁺ ion detection.

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Fig. 4a shows the color change towards different ions of PNF-AuNPs solution. 50 μL of various ions’ solution samples were added into 200 μL of PNF-AuNPs samples, and then the color change of the solutions after 5 min was observed and captured. Besides Hg²⁺ made the solution turned deep purple, both Cd²⁺ and Cu²⁺ also turned a little blue and grey, which may be caused by the similar ion-induced aggregation mechanism reported previously (Zhang et al., 2012). However, the color change was not as remarkable as that caused by Hg²⁺. The response of PNF-AuNPs towards various ions was compared by UV–Vis spectra (Fig. 4b). It can be found that there is no countable response towards other ions except Hg²⁺. Experimental results indicate that with adding Hg²⁺ ions, the peak at 525 nm decreased, while a new peak at 300 nm appeared. It suggested the interaction of Hg²⁺ and Au may occur in the solution. For comparing the sensitivity and selectivity towards Hg²⁺, ratio of peak values of 300 nm (I₃₀₀) and 525 nm (I₀) was compared and demonstrated in Fig. 4c. Compared with control group (red bar), only Hg²⁺ caused a remarkable rising of I₃₀₀/I₀, which demonstrated excellent selectivity to Hg²⁺.

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Fig. 4 Selectivity experiments of PNF-AuNP-based colorimetric Hg²⁺ biosensor. (a) Color change of PNF-AuNPs probes after adding various metal ion samples, including Hg²⁺, Pb²⁺, Co²⁺, Fe²⁺, Cu²⁺, Cd²⁺, Mg²⁺, Zn²⁺, Ca²⁺, and blank sample, respectively. The photo was adjusted just for reaching the colors that were observed by bare eyes. (b) UV–Vis spectra of samples that added 50 μL ions and placed for 5 min. There was a new peak appeared in Hg²⁺ sample, at the wavelength of 300 nm. (c) Ratio of peak value at 300 nm and 525 nm (I₃₀₀/I₀) of each sample in (b), the red bar refers to the blank group. (d) Color change of PNF-AuNPs towards series concentrations of Hg²⁺. An aggregation occurred when the concentration reached 50 μM, (e) Responding results of PNF-AuNPs towards Hg²⁺ samples, which were by UV–Vis spectra. The peak intense of PNF-AuNPs at about 525 nm decreased with the increasing of Hg²⁺ (0–70 μM), while the peak at about 300 nm showed up and increasing with the increasing of Hg²⁺. (f) Linear-fit curve of concentration-I₃₀₀/I₀ relationship, R² = 0.991.

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In order to establish a linear response of the concentrate-color relation, detection of series concentrations Hg²⁺ samples was performed. The further colorimetric result shows that the aggregation occurred with adding Hg²⁺ solution (2, 10, 15, 30, and 50 μM, respectively) into PNF-AuNPs samples, leading to obvious color change (Fig. 4d), which agreed well with the UV–Vis spectra results, and revealed a potential ability of visual detection. Change of color indicates change of aggregation, and suggests the formation of heavily aggregation and entanglement of PNF-AuNPs, which is observed in SEM and TEM images (Figs. S5 and S6). Since Hg²⁺ have strong interactions with peptide nanofiber, some AuNPs seem fall off from the peptide nanofibers after adding the Hg²⁺ sample. The aggregation of PNF-AuNPs was also measured by UV–Vis spectra (Fig. 4e). The peak intensity increases of 300 nm with the increasing of Hg²⁺ concentration, while the peak at 525 nm decreased. The ratio of peak intensity at 300 nm (I₃₀₀) and the peak intensity of PNF-AuNPs (I₀) exhibit a linear change along the increasing of Hg²⁺ concentration (Fig. 4f). The created PNF-AuNPs reached a linear detection of Hg²⁺ in a concentration range of 10–70 μM (R² = 0.991), and the detection limit was calculated to be 0.21 μM (S/N = 3). This colorimetric response towards Hg²⁺ proves that PNF-AuNPs possess the ability of monitoring Hg²⁺ and other heavy metal ions in water sample.

3 Experimental

4 Conclusions

In this work, we demonstrated a green and efficient self-assembly strategy for electrochemical and colorimetric sensor, based on fibril architecture of peptide-AuNPs. The PNF-AuNP nanofibril exhibited excellent sensing performances for electrochemical and colorimetric detections, with good sensitivity, selectivity, and reproducibility. The method of fabricating peptide-induced AuNPs offers a versatile platform for biomineralization and assembly of inorganic nanoparticles and nanoclusters. Combining biocompatibility and highly sensitivity of PNF-AuNPs, this peptide-nanoparticle architecture would be promising candidates for biosensing, cell imaging, biomarker, and other biomedical applications in the future.