Photochemical induced formed Au nanomaterial with size and shape controlled by luminol–pepsin chemiluminescence reaction

Abstract

Size and shape controlled Au nanomaterials (AuNMs) were generated in different alkaline luminol–pepsin (Pep) chemiluminescence (CL) reaction solutions based on the photochemical induced effect of luminol. These findings showed that luminol photochemical intensity could be used as a major control parameter for metal NMs growth.

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Size and shape provides important and profound influence over several physical and chemical properties of nanomaterials (NMs), including luminescence,^1–5 conductivity,^6–8 and catalytic activity.^9–11 The preparation of the NMs with controlled size and shape is an attractive and paramount goal in developing and fabricating all types of biosensors and bioelectronics in the field of chemistry,^12,13 materials,¹⁴ biology,¹⁵ and medicine.¹⁶ The method for simultaneously controlling both size and shape remains an extremely difficult task, which is always complicated and time consuming, and is only partially characterized by the interplay of kinetics and thermodynamics.¹⁷ Recently, tremendous efforts have been made for regulating the properties of the NMs by controlling quantum confinement phenomena (QCP) in the process of NMs growth.^18–21 The nature of QCP for photochemical reactions is mainly from the considerably active free radical, which was associated with electronic transitions from excited state to ground state.^22–26 Based on this, photo-induced technology for forming NMs with controlled size and shape could shift the apparent thermodynamic equilibrium of redox reactions to favor room temperature catalysis with mild reaction conditions.^27–30 However, the nature of time-consumption for the formation of NMs is not completely changed due to the shortage of special endogenous power in a wide spectral range. Photochemical induced technology^31,32 enables the characterization of endogenous fluorescence and elimination of photo-bleaching, particularly associated with strong QCP in the photochemical process. Considering the novel characterization for photochemical induced technology, it could explore a new method for forming NMs with controllable size and shape under room temperature and mild conditions.

Luminol, as one of common organic photochemical induced agents (E_m = 425 nm), has a different quantum efficiency with alkaline medium-dependent nature in the luminol chemiluminescence (CL) reaction.³³ In a previous report, HAuCl₄ was effectively catalysed to form Au nucleation and eventually grown nanoparticles (AuNPs),^34,35 which could accelerate the electronic transition rate and enhance the luminol CL intensity in the luminol–HAuCl₄ CL reaction.^34,36 In our former work, we found that HAuCl₄ and pepsin (Pep) sharply enhanced the CL intensity under alkaline conditions (pH = 10.5) (support Fig. 1) and formed the Au nanoclusters (AuNCs), changing the colour of the reaction solution rapidly from light yellow to light red. The possible mechanism might be attributed to the joint action of the luminol photochemical induced effect and micro-change of Pep conformation in the CL system under alkaline conditions.

Fig. 1 Scheme of different sized and shaped AuNMs formed in the photochemical induced luminol–Pep CL system with E_m = 425 nm.

Hence, here, we report a native luminol photochemical induced method for forming mono-disperse Au nanomaterials (AuNMs) with sizes from 1 to 400 nm, such as spherical-like AuNCs, flower-like AuNPs and regular hexagonal-like Au nanosheets (AuNSs), in CL reaction solution at different pH (Fig. 1). All the above mentioned sizes of AuNMs were rapidly formed in a CL reaction solution for 30 min based on the luminol photochemical induced at E_m = 425 nm. In this approach, according to the generation mechanism for protein protected AuNMs by Glomm's group,³⁷ combined with the instability conformation of Pepsin in alkaline solution³⁸ and different quantum efficiency with alkaline medium-dependent nature in luminol CL reaction,³³ the possible mechanism for the controlled size and shape of AuNM in Pep–luminol alkaline solution with different pH might be attributed to the following: first was the nucleation stage. Pep surface contain massive negative charges weakly acidic or neutral conditions (above Pep's pK_a), and charge density matching between Au³⁺ and negatively charged amino acid residues yields high metal concentrations, forming the Pep–Au³⁺ complex on the surface of Pep.³⁹ This step might provide the necessary microenvironment for triggering Au nucleation growth with high redox potential. Second was initialization and growth stage. Based on the property of high quantum efficiency of luminol at alkaline solution, the electron transferring rate between Pep–Au³⁺ complex could be accelerated by the excited 3-aminophthalate, which resulted in the formed of free radical with light emission at different pH luminol alkaline solution. It was worth noting that Pep as an acidic protein, has the instability conformation and construction at the alkaline solution. It was attributed to the interaction of Asp11 and Asp159 in Pep construction, which showed abnormally high pK_a values compared with other amino acid residues, such as Glu4, Glu13 and Asp118, with an abnormally low pK_a value.³⁹ For spherical-like AuNCs, Au³⁺ on the surface of Pep flowed into the vicinity of negatively charged amino acid residues due to the principle of charge density matching, and eventually formed pepsin protected AuNCs with the bond of Au–S in the cavity of pepsin at a pH of 10.5. At a pH of 12.0, Pep had instability conformation at the alkaline solution. The instability conformation and partial unfolding of the polypeptide chain of Pep resulted in close contact between adjacent Au nucleation and subsequent aggregation on the Pep surface, eventually forming AuNPs with irregular shape, described as flower-like AuNPs. When the pH reached to 14.0, the instability conformation of Pep increased, and the collision between Peps further exacerbated that led to a high contact probability between exposed Peps. Under this situation, the conformation flexibility of the polypeptide chain, the charge density surrounding nucleation sites and the proximity of the nucleation to the surrounding aqueous environment could induce Au nucleation to orderly aggregate with luminol photochemistry and eventually form hexagonal-like AuNSs. The last was the termination stage. Termination could occur via the depletion of quantum efficiency of luminol produced by QCP, decrease in redox potential (dynamics) and equilibrium of Au³⁺ concentration between the bulk phase and Pep surface (electrostatic and steric). At this stage, the colour of the solution remained almost unchanged; the electrostatic barrier for bringing the Au atom into close proximity becomes too large to overcome, thus preventing further growth. For the above mentioned process, the micro-change of Pep confirmation acted as the template, which could provide the micro-environment for Au nucleation accumulation and aggregation, ultimately accelerating the growth tendency of Au nanocrystals in the CL reaction for forming different sizes and shapes of AuNMs in the alkaline solution. With the joint action of the QCP by the photochemical induced and the micro-change of Pep conformation mediated by the nature of alkaline conditions, different AuNMs were eventually formed at different CL intensity. As a control experiment, there were only unified and mono-dispersed quasi-spherical structured AuNPs formed with average diameters of 55 ± 4.2 nm for different pH in the absence of Pep (support Fig. 2). Throughout the entire process, we could speculate that pH, as the key factor, not only influenced the CL intensity, which promoted a rapid reaction by photochemical induced mechanism, but also induced the instability conformation or construction of Pep in alkaline solution, which could provide the effective template or microenvironment for Au atom accumulation and aggregation formed with the different size and shape controlled AuNMs in alkaline solution.

Fig. 2 (a) TEM image of AuCNs (1–2 nm), AuNPs (35 ± 5.6 nm) and AuNSs (400 ± 25 nm). (b) EDX energy. Spectrum of AuNMs.

The different sized and shaped AuNMs could be controlled by the sideway of different CL intensity at different pH values. Interestingly, on increasing pH, the CL intensity showed a decreasing tendency; on the contrary, the average particle size showed an increasing tendency. For example (Fig. 2a), injecting luminol solution with a pH of 10.5 obtained 1–2 nm spherical-like AuNCs associated with a CL intensity of 1150 pH 12.0, yielding 35 ± 5.6 nm flower-like AuNPs for a CL of 125, whereas pH 14.0 produced 400 ± 25 nm regular hexagonal-like AuNSs for a CL of 32. The above mentioned results fit the hypothesis that alkaline luminol solution not only influenced the QCP but also induced the instability conformation of Pep. In addition, on increasing the pH of alkaline solution, the CL intensity measured by PMT showed exponential decay trends. These trends would be partially relative to the pH influenced by the CL intensity but are mainly subjected to reducing the quantum effect and enhancing the scatter effect, while the AuNMs particle size increase. The stability of the AuNMs colloid solution formed in the luminol–Pep CL reaction was also investigated. With time, protein protected AuNCs showed good stability for 48 days; the flower-like AuNPs have moderate stability for 15 days and hexagonal-like AuNSs have relatively poor stability for only 7 days. The stability of AuNMs in colloid solution mainly depended on particle size, adulterating degree and surface ligand.^40,41 The XRD spectrum (Fig. 2b) showed that AuNMs prepared in luminol–Pep CL reaction had virtually no adulterating element with only Au, Na, C, O and Cl. From the viewpoint of particle size and surface ligand, different sized and shaped AuNMs had different stability in alkaline solution. Spherical-like AuNCs were capped into the Pep construction with the Au–S bond with a hydrophilic surface. This characterization of AuNCs endowed it good stability in alkaline solution. Flower-like AuNPs had larger specific surface area because of conformation instability and partial unfolding of the polypeptide chain, which could provide the site for Au nucleation aggregating on the surface of Pep. Increasing particle size and decreasing hydrophilic group enables AuNPs to have moderate stability in alkaline solution compared with the long stability of AuNCs. Regular hexagonal-like AuNSs' shape and size mainly depend on the collisions of inter-Peps and polypeptide chain folding. The relative poor stability was attributed to the maximum particle size and surface tension of strong alkaline solution.

These results indicated that luminol, as the photochemical induced agent, could rapidly induce Au³⁺ to form Au nucleation and eventually grow different sized and shaped AuNMs. As expected, the UV-vis results (Fig. 3a) showed that the absorption peaks of Pep and HAuCl₄ were 265 nm and 280 nm, respectively, which was smothered by the characterization absorption peaks of luminol at 285 nm and 325 nm. The characteristic absorption peak of luminol exhibited a decreasing tendency, while the pH increased from 10.5 to 14.0. An absorption peak in the 525–560 nm range was the characteristic absorption peak of Au with the tendency of blue shifting in the pH of 10.5–14.0, which could be attributed to the more luminol derived, 3-AP absorbed on the Au surface with the bond of Au–N. However, interestingly, the colour of the AuNCs solution was light red, while the pH was set at 10.5. If pH was set to 12.0, the solution was red, which then turned to dark red when pH was increased to 14.0 (Fig. 3b); these results corresponded with the UV-vis results.

Fig. 3 (a) UV-vis spectrum of luminol–HAuCl₄–pepsin reaction with pH from 10.0 to 14.0. The corresponding concentrations of pepsin (1), HAuCl₄ (2) and luminol (3) were 1.0 × 10⁻⁵, 2.5 × 10⁻⁵ and 2.5 × 10⁻⁴ mol L⁻¹, and (4–9) were the different pH of 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5 and 14.0, respectively. (b) The different color of AuNMs produced with different pH from 14.0 to 9.0 (A–I) in 30 min.

In conclusion, we found that with the joint action of the QCP by the photochemical induced and the micro-changed Pep conformation mediated alkaline luminol solution, the different AuNMs with controlled size and shape could be formed in the CL reaction solution. This demonstrated that alkaline luminol solution not only influenced the QCP of CL reaction but also acted as the micro-environment of Pep conformation at room temperature. Upon further study, this unusual and intriguing feature would helpful in clarifying some photo-induced mechanisms of AuNMs formation in different CL reactions, which presently remain obscure. For practical purposes, this procedure opened a simple and fast way to tailor the properties of photochemically prepared metal NMs for specific applications by their size and shape in the fields of biosensor and bioelectronics.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21275118) and the Open Fund from Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07283a

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