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. 2020 Sep 15;5(38):24487–24494. doi: 10.1021/acsomega.0c02956

Fluorescence Quenchers Manipulate the Peroxidase-like Activity of Gold-Based Nanomaterials

Yu-Shan Chen 1, Zhi-Wen Chen 1, Yu-Wen Yuan 1, Kuan-Chung Chen 1, Ching-Ping Liu 1,*
PMCID: PMC7528283  PMID: 33015465

Abstract

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Although the regulation of the enzyme-like activities of nanozymes has stimulated great interest recently, the exploration of modulators makes it possible to enhance the catalytic performance of nanozymes, though doing so remains a big challenge. Herein, we systemically studied the effects of fluorescence quenchers on the peroxidase-like activity of bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) based on photoinduced electron transfer (PET). We found that PET quenchers can not only quench the fluorescence of BSA-AuNCs but also regulate their intrinsic peroxidase-like activity. Importantly, both BSA and human serum albumin (HSA) could enhance the peroxidase-like activity of Cu2+, which provided a new sensing platform for distinguishing BSA and HSA from other thiol-containing biomolecules. The PET quenchers could also manipulate the peroxidase-like activity of polyvinylpyrrolidone-stabilized gold nanoparticles (PVP-AuNPs), which exhibited some opposite results between PVP-AuNPs and BSA-AuNCs. The opposite effects on BSA-AuNCs and PVP-AuNPs were speculated to highly depend on their surface properties. Our findings offer an efficient strategy for tuning the peroxidase-like activities of gold-based nanozymes.

Introduction

Nanomaterials capable of functions similar to those of natural enzymes (i.e., nanozymes) have stimulated much research interest because of their striking features, such as low cost and robustness.1,2 Currently, nanozymes are known to have various enzyme-like characteristics, such as those of peroxidase, oxidase, glucose oxidase, catalase, and superoxide dismutase.2 Typically, when nanozymes can catalyze hydrogen peroxidase (H2O2) oxidizing substrates and yielding products similar to those observed in peroxidase-catalyzed reactions, such nanozymes possess the peroxidase-like activity. Nanozymes with such activity have been widely used in the fields of biosensing and immunoassay, leading to the formation of a large family.3 On the basis of the catalytic cycle of horseradish peroxidase (HRP), the mechanism for nanozymes has been proposed to involve two major processes.4 One is the electron transfer processes among nanozymes, H2O2, and peroxidase substrates;5 the other is the formation of hydroxyl radicals (•OH), which act as the key intermediate during the peroxidase mimic reactions.6 However, the exact mechanisms of nanozymes may depend on the natures of different nanomaterials.

Recently, fluorescent metal nanoclusters with an ultrasmall size (<2 nm) have exhibited great promise as optical reporters in fluorescence-based sensing techniques for elucidating photophysical science, and thus they hold potential as research tools. In addition to their fluorescence, the catalytic properties of metal nanoclusters are also attractive because of their size-specific electronic and geometric structures.7 Among the various metal nanoclusters, bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) were first reported to possess excellent peroxidase-like activity because of their small size, high surface-to-volume ratio, and strong affinity to the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB).8 Subsequently, a dual fluorescence and colorimetric sensor was developed because dopamine (DA) can not only quench the fluorescence of BSA-AuNCs via photo-induced electron transfer (PET) but also inhibit their intrinsic peroxidase-like activity.9 That work has led to the intriguing issue of whether fluorescence quenchers may affect the peroxidase-like activity of BSA-AuNCs. To date, the fluorescence-quenching mechanisms of BSA-AuNCs have been widely studied10 and can be separated into two categories: one involving the aggregation and etching of the main Au component,11,12 and the other relying on the PET and fluorescence resonance energy transfer (FRET) between BSA-AuNCs and quenchers.13,14 It is straightforward that aggregation and etching of BSA-AuNCs inevitably diminish their peroxidase-like activity because of direct loss of surface accessibility (i.e., active sites). Comparatively, the influences of PET and FRET quenchers on the peroxidase-like activity of BSA-AuNCs are obscure because no chemical interaction takes place between such quenchers and BSA-AuNCs without irradiation. PET pairs can facilitate electron transfer from the donor to the acceptor based on their redox potentials, and then the energy in the excited states is dissipated nonradiatively.15 The efficiency of the PET process is mainly controlled by the spatial distance between the donor and the acceptor, the relationship between excitation and transfer processes, the free energy of the reaction, and the polarity of solvents.15−17 It remains unclear whether such catalytic properties of fluorescent nanozymes can be manipulated by their corresponding fluorescence quenchers. Accordingly, BSA-AuNCs are appropriate for investigating the effects of PET quenchers on their peroxidase-like activity.

In this work, we systemically studied the effects of PET quenchers on the peroxidase-like activity of BSA-AuNCs. Six quenchers including Hg2+, Cu2+, DA, ascorbic acid (AA), 3,3′-diethylthiatricarbocyanine iodide (DTTC I), and nitrite ions (i.e., NO2–) were chosen because all of them could quench the fluorescence of BSA-AuNCs via the PET process.10 The possible mechanism to manipulate the peroxidase-like activity of BSA-AuNCs by PET quenchers was explored and discussed. In addition, the PET quenchers were also applied to regulate the peroxidase-like activity of AuNPs. Our findings offer an efficient strategy for tuning the peroxidase-like activities of gold-based nanozymes and even make it possible to enhance catalytic performance, which remains a big challenge.

Results and Discussion

Peroxidase-like Activity of BSA-AuNCs with Fluorescence Quenchers

The BSA-AuNCs were prepared using BSA as a template and reducing agent according to a previous report.18 The transmission electron microscopy (TEM) images, UV–vis absorption, and fluorescence spectra of BSA-AuNCs were obtained as shown in Figure S1, and all were consistent with the previous report.18Figure 1A shows that these PET quenchers could quench the fluorescence of BSA-AuNCs under UV light (at 254 nm) irradiation. Subsequently, the peroxidase-like activities of BSA-AuNCs in the absence and presence of these quenchers were determined based on the oxidation of the peroxidase substrate (i.e., TMB) to generate the blue product (i.e., the oxidized TMB). Because BSA-AuNCs possessed the intrinsic peroxidase-like activity, the colorless TMB turned blue in the H2O2/TMB system.8 Interestingly, the additions of Hg2+, DA, and AA could inhibit the peroxidase-like activity of BSA-AuNCs, whereas Cu2+, DTTC I, and NO2– could not. In other words, fluorescence quenching did not necessarily result in the inhibition of the peroxidase-like activity of BSA-AuNCs. Figure 1B shows the behaviors of these PET quenchers alone in the H2O2/TMB system. Among them, Cu2+, DTTC I, and NO2– could cause oxidation of TMB to produce a blue color with the major absorbance around 652 nm (i.e., the oxidized TMB). In addition, only NO2– could directly interact with TMB in the absence of H2O2 (Figure 1C). It should be noted that DTTC I exhibited a weak absorption feature over 600–750 nm, which was distinguishable from the oxidized TMB with the absorption band around ∼650 nm. Cu2+ has been reported to possess the peroxidase-like activity,20 which was consistent with our observation (Figure 1B). As for DTTC I, its influence on the peroxidase-like activity of BSA-AuNCs will be discussed below. The quencher NO2– was excluded from the following experiments because of the interference of the peroxidase-like activity. These observations indicated that PET quenchers can not only quench the fluorescence of BSA-AuNCs but also regulate their intrinsic peroxidase-like activity.

Figure 1.

Figure 1

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(A) Fluorescence quenching (upper) and the corresponding peroxidase-like activities (lower) of BSA-AuNCs in the absence and presence of six quenchers (Hg2+, Cu2+, DA, AA, DTTC I, and NO2–), respectively. The absorption spectra of the oxidized TMB (∼650 nm) produced when adding various quenchers in the presence of (B) H2O2 and TMB and (C) TMB only, respectively. Experiments were performed with BSA-AuNCs (5 μM) in acetate buffer (0.1 M, pH 4.0) and H2O2 (50 mM) with TMB (0.2 mM) as the peroxidase substrate.

The peroxidase-like activities of BSA-AuNCs inhibited by DA, Hg2+, and AA (Figure 1A) were the same as reported previously.9,21,22 The inhibition mechanism of Hg2+ has been ascribed to the active site of Au+ on the surface of BSA-AuNCs being blocked by Hg2+ through the formation of metallophilic bonding.21 The peroxidase-like activity stems from the ability to decompose H2O2 into •OH, which was the key intermediate for the oxidation of peroxidase substrates.6 AA and DA are both antioxidants,23,24 which can efficiently react with reactive oxygen species such as •OH and H2O2 to prevent the oxidation of peroxidase substrates, thereby diminishing the peroxidase-like activity of BSA-AuNCs. Therefore, if the PET quenchers were antioxidants or interacted with active sites, the peroxidase-like activity of BSA-AuNCs could be inhibited effectively.

Enhancement of the Peroxidase-like Activity of BSA-AuNCs

Although the additions of Cu2+ and DTTC I quenched the fluorescence of BSA-AuNCs, they did not inhibit the corresponding peroxidase-like activity (Figure 1A). In contrast, an apparent increase in absorbance at 652 nm was observed with greater additions of Cu2+ into the BSA-AuNCs solution (Figure 2A), indicating that the peroxidase-like activity was enhanced by Cu2+. Previous work revealed that Cu2+ could bind to BSA,25 leading to fluorescence quenching of BSA-AuNCs through electron transfer. It was speculated that Cu2+ preferentially interacted with the outside BSA rather than with the interior AuNCs; if so, the peroxidase-like activity could be attributed to Cu2+ and BSA-AuNCs simultaneously, leading to an increase in the catalytic performance of BSA-AuNCs. Another notable feature was that the mixture of BSA and Cu2+ significantly enhanced the peroxidase-like activity of Cu2+ by more than 50%, although BSA did not exhibit the peroxidase-like activity (Figure 2B). In addition, the enhancement factor was equivalent when human serum albumin (HSA) was used to replace BSA because HSA is structurally similar to BSA26 and Cu2+ also has a strong affinity with HSA.25 Moreover, the similar affinities between Ni2+ and either BSA or HSA led to no enhancement of the catalytic activity because Ni2+ itself did not behave as a peroxidase mimic. Figure 2C displayed that the addition of BSA and HSA could both enhance the peroxidase-like activity of Cu2+ significantly, but other protein thiols such as trypsin and lysozyme could not. Cu2+ was recognized to have a high affinity with histidine because histidine typically exists in the protein chain.25,27 The surface functionalization of histidine on Fe3O4 nanoparticles has also been reported to mimic the active site of natural peroxidase enzyme (i.e., HRP), thereby enhancing the peroxidase-like activity of Fe3O4.28 However, the addition of histidine into Cu2+ only slightly increased the peroxidase-like activity of Cu2+ (Figure S2B), indicating that the interaction of Cu2+ with histidine did not play the key role. It was speculated that other amino acids present in BSA or the structural coordination between Cu2+ and BSA (or HSA) might affect the peroxidase-like activity of Cu2+. These results indicated that such unexpected promotion of the catalytic activity of Cu2+ provided the specificity of the sensing platform for distinguishing either BSA or HSA (Figure S3) from other biological thiols.29,30

Figure 2.

Figure 2

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Examination of the peroxidase-like activity of BSA-AuNCs based on the absorption spectra of the oxidized TMB with a major absorbance peak at 652 nm. Change in the peroxidase-like activity of BSA-AuNCs was measured by increasing the concentrations of (A) Cu2+ and (D) DTTC I, respectively. (B) Peroxidase-like activities of Cu2+ and Ni2+, measured in the presence of BSA and HSA, respectively. (C) Relative absorbance at 652 nm for Cu2+ in the absence and presence of different protein thiols (BSA, HSA, trypsin, and lysozyme), respectively. The absorption spectra of oxidized TMB (E) and their corresponding absorbance at 652 nm (F) responsible for the peroxidase-like activity of BSA-AuNCs in the absence and presence of NaI, KI, and DTTC I, respectively.

Figure 2D shows that the peroxidase-like activity of BSA-AuNCs was increased by adding DTTC I. DTTC I is a quaternary ammonium salt and thus can dissociate I– ions in aqueous solution.29 I– ions have been reported to catalyze the oxidation of TMB in the presence of H2O2,30 and DTTC I was also observed to catalyze the TMB/H2O2 system (see Figure 1C). Accordingly, the enhancement mechanism was considered to be highly associated with I– ions from DTTC I. To verify this possibility, DTTC I was replaced with NaI and KI as the source of I– ions. As shown in Figure 2E,F, the peroxidase-like activity of BSA-AuNCs was significantly increased when BSA-AuNCs were premixed with either NaI or KI in the H2O2/TMB system. In addition, the fluorescence of BSA-AuNCs could not be quenched by I– ions when adding either NaI or KI into the solution of BSA-AuNCs (Figure S4). Therefore, the enhancement of the peroxidase-like activity of BSA-AuNCs by DTTC I resulted from the corresponding counter ions of I– in DTTC I. This finding indicated that the chemical reaction between I– ions and TMB was much more dominant than the PET relation between BSA-AuNCs and the cyanine dyes.31

Peroxidase-like Activity of BSA-AuNCs Mediated by Light Irradiation

The PET process takes place upon irradiation, which may contribute to the peroxidase-like activity of BSA-AuNCs in the presence of their PET quenchers. It should be noted that the fluorescence quenching of BSA-AuNCs by PET quenchers was typically achieved at neutral pH under UV light excitation (at 254 or 365 nm).10 However, the peroxidase-like activity should be examined under an acidic condition (∼pH 4), and H2O2 was easily photolyzed by UV light to produce •OH, resulting in the inference of the peroxidase-like activity. In addition, DTTC I and NO2– should be excluded from the experiments upon irradiation because they can oxidize TMB in the presence of H2O2 (Figure 1B). For this purpose, the peroxidase-like activity of BSA-AuNCs with PET quenchers under an acidic condition (at pH 4) was investigated without and with visible light irradiation (as shown in Figure S5). Figure 3A shows that the peroxidase-like activity of BSA-AuNCs was significantly enhanced upon visible light irradiation, which might be attributable to the oxidase-like activity of BSA-AuNCs by visible light activation.19 However, the addition of Hg2+, Cu2+, DA, and AA did not benefit the peroxidase-like activity of BSA-AuNCs during visible light irradiation (Figure 3B). Similarly, Hg2+, DA, and AA still predominantly inhibited the catalytic performance of BSA-AuNCs, and Cu2+ seemed harmful to the effect of irradiation. It should be noted that the fluorescence quenching of BSA-AuNCs by Cu2+ could still take place at pH 4 (as shown in Figure S6). A previous work reported that hot electrons generated by irradiating laser (at 532 nm) were recognized as the key factor in the enhancement of the peroxidase-like activity of AuNPs.32 It was speculated that hot electrons might preferentially transfer to PET quenchers such as Cu2+, thereby reducing the enhancement effect of irradiation on the peroxidase-like activity of BSA-AuNCs.

Figure 3.

Figure 3

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(A) Absorbance at 652 nm of the oxidized TMB changed with increasing time for BSA-AuNCs with light on and off, respectively. (B) Relative absorbance at 652 nm for BSA-AuNCs in the absence and presence of quenchers with light on (the white bar) and off (the black bar), respectively.

Extension from AuNCs to AuNPs

Because these PET quenchers could manipulate the peroxidase-like activity of BSA-AuNCs, it was proposed that they might function similarly when gold nanoparticles (AuNPs) were used instead of BSA-AuNCs. To investigate this possibility, polyvinylpyrrolidone-stabilized AuNPs (PVP-AuNPs) were prepared33 as shown in Figure S7 because of their high stability34 and intrinsic peroxidase-like activity.35Figure 4 presents the peroxidase-like activities of PVP-AuNPs in the absence and presence of these PET quenchers as stated above. The catalytic activity of PVP-AuNPs was retained by the addition of Hg2+ and Cu2+ but inhibited by DA, AA, and DTTC I. Interestingly, the effects of Hg2+ and DTTC I on PVP-AuNPs were completely opposite to those on BSA-AuNCs. Because the PVP-AuNPs (∼10 nm) were relatively large and easy to separate from quenchers by centrifugation, whether the regulation of the catalytic activity by quenchers was reversible could be examined. More significantly, the peroxidase-like activity of PVP-AuNPs was almost recovered after centrifugation to remove AA, Hg2+, and Cu2+, respectively, indicating that no chemical reactions occurred between the AuNPs and quenchers. Unlike BSA-AuNCs, Hg2+ had no influence on the peroxidase-like activity of PVP-AuNPs, similar to citrate-stabilized AuNPs in the presence of Hg2+.36 Because Hg2+ could be reduced to Hg0 by either citrate ions or the reducing agent NaBH4, it was considered that PVP-AuNPs might adsorb BH4– on the surface of AuNPs37 and then Hg2+ was preferentially reduced to Hg0. However, in the preparation of BSA-AuNCs, BSA was typically used to replace NaBH4 as the reducing agent. Accordingly, Hg2+ might favor blocking the active site of Au+ on BSA-AuNCs rather than being reduced to Hg0. To further confirm this, X-ray photoelectron spectroscopy (XPS) was used to verify the change of Au+ for PVP-AuNPs and BSA-AuNCs in the absence and presence of Hg2+, respectively. Figure S6 shows the XPS spectra of Au 4f5/2 and 4f7/2, respectively. Each band could be deconvoluted into two distinct components of Au0 and Au+,35,38 and the percentage of surface Au+ was thereby calculated based on the peak areas of Au+ and Au0. Obviously, the addition of Hg2+ did not change the percentage of Au+ on PVP-AuNPs (Figure S8A), whereas it did change that on BSA-AuNCs (Figure S8B). Moreover, the consumption of Hg2+ by NaBH4 could retard the inhibition of the peroxidase-like activity of BSA-AuNCs (Figure S9). Taken together, although the detailed mechanism of PVP-AuNPs against Hg2+ blocking active sites was still unclear, these results indicated that whether Hg2+ is reduced or not could regulate the peroxidase-like activity of AuNPs and AuNCs.

Figure 4.

Figure 4

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Peroxidase-like activity of PVP-AuNPs in the absence and presence of five quenchers (Hg2+, Cu2+, DA, AA, and DTTC I) before (upper) and after centrifugation (lower), respectively. The red boxes mark the two groups that are different from those for BSA-AuNCs (Figure 1A).

In addition to Hg2+, the effect of DTTC I on PVP-AuNPs also exhibited the opposite result from BSA-AuNCs. According to the literature, I– ions inhibited the peroxidase-like activity of casein-stabilized AuNPs because of the formation of the Au–I bond.39 Similarly, the peroxidase-like activity of PVP-AuNPs could be inhibited by I– ions from DTTC I, KI, and NaI, respectively (Figure S10). In addition, the removal of DTTC I did not lead to the recovery of the catalytic activity of PVP-AuNPs (Figure 4). These observations indicated that DTTC I decreased the peroxidase-like activity of PVP-AuNPs because I– ions blocked active sites via the formation of the Au–I bond.39 The next question was why I– ions could not diminish the peroxidase-like activity of BSA-AuNCs. Because I– ions could not penetrate the interior of the BSA matrix,40,41 it was speculated that free I– ions predominantly reacted with TMB and H2O2, rather than blocking active sites on BSA-AuNCs. Therefore, the opposite effects of Hg2+ and DTTC I on BSA-AuNCs and PVP-AuNPs further demonstrated that the peroxidase-like activities of AuNPs and AuNCs were highly dependent on their surface properties. Our findings could offer efficient strategies for tuning the peroxidase-like activities of gold-based nanomaterials.

Conclusions

In this work, we systemically studied the effects of PET quenchers on the peroxidase-like activity of BSA-AuNCs. Our results showed that fluorescence quenching of BSA-AuNCs by their PET quenchers did not necessarily correspond to the inhibition of their peroxidase-like activity. Impressively, the peroxidase-like activity of BSA-AuNCs was increased by Cu2+ and DTTC I, respectively. Because of the specificity of Cu2+ binding to BSA rather than to AuNCs, the combination of Cu2+ and BSA-AuNCs could act as two sources of peroxidase mimics. Unexpectedly, both BSA and HSA could enhance the peroxidase-like activity of Cu2+, which provided a good sensing platform for distinguishing BSA and HSA from other biothiols. As for DTTC I, enhancement of the peroxidase-like activity of BSA-AuNCs resulted from the corresponding counter ions of I– rather than from the cyanine dye. In addition, the peroxidase-like activity of BSA-AuNCs with PET quenchers was also investigated without and with visible light irradiation, but the presence of PET quenchers decreased the enhancement effect of irradiation on the catalytic activity. It was speculated that hot electrons might preferentially transfer to PET quenchers such as Cu2+, thereby reducing the enhancement effect of irradiation on the peroxidase-like activity of BSA-AuNCs. Moreover, the PET quenchers could also manipulate the peroxidase-like activity of PVP-AuNPs, which exhibited some opposite results between PVP-AuNPs and BSA-AuNCs. The opposite effects of Hg2+ and DTTC I on BSA-AuNCs and PVP-AuNPs further demonstrated that the peroxidase-like activities of AuNPs and AuNCs were highly dependent on their surface properties. Our findings offer an efficient strategy for tuning the peroxidase-like activity of gold-based nanozymes and even make it possible to enhance catalytic performance, which typically is relatively difficult to achieve.

Experimental Section

Chemicals and Reagents

BSA, HSA, hydrogen tetrachloroaurate(III) tetrahydrate (HAuCl4·3H2O), sodium hydroxide (NaOH), TMB, DA, AA, mercury(II) chloride (HgCl2), copper(II) chloride (CuCl2), sodium nitrite (NaNO2), nickel(II) nitrate hexahydrate (Ni(NO3)2·6H2O), and DTTC I were all purchased from Sigma. All reagents were used without further purification.

Preparation of BSA-AuNCs

BSA-AuNCs were prepared according to a previous report18,19 with minor modification. Briefly, the aqueous HAuCl4 solution (5 mL, 10 mM) was added into the BSA solution (5 mL, 50 mg mL–1) in a 37 °C water bath with stirring for 30 min. Subsequently, the NaOH (0.5 mL, 1.0 M) solution was added to the mixture under 37 °C incubation with vigorous stirring. The color of the mixture changed from pale yellow to deep brown, and the resulting solution was continuously stirred for 12 h. BSA-AuNCs were obtained after centrifugation at 6000 rpm with centrifugal filters (Ultracel-3K) to remove excess reactants and washed with deionized (DI) water at least three times. The final solutions were stored at 4 °C in a refrigerator for further experiments.

Preparation of PVP-AuNPs

The preparation of PVP-AuNPs followed a seed-mediated growth method that allows easy control of the size of AuNPs.33 All glassware and stir bars were cleaned in aqua regia (1:3 HNO3/HCl) and rinsed with DI water before use for the preparation of PVP-AuNPs. Initially, 555 mg of PVP (molecular weight 10 kDa) was added into an aqueous solution of HAuCl4 (1 mM, 50 mL), and the mixture was stirred at 4 °C for 30 min. Subsequently, 5 mL NaBH4 (100 mM) was rapidly added into the mixture under vigorous stirring. After stirring at 4 °C for another 30 min, the resulting solution was kept as seeds for preparation of PVP-AuNPs. Next, 1 mL HAuCl4 (50 mM) was diluted by DI water (35 mL), 555 mg PVP was added, and the solution was stirred at 4 °C for 30 min. The above solution was added to 10 mL of the seed solution, and the mixture was stirred for another 30 min. After that, 15 mL AA (5 mM) was slowly dropped into the above mixture and the solution was stirred at 4 °C for 2 h. Finally, PVP-AuNPs were obtained after centrifugation at 10,000 rpm to remove excess reactants and washed with DI water at least three times. The solution of PVP-AuNP was stored at 4 °C in a refrigerator for further experiments.

Characterization

UV–vis absorption spectra were obtained using a JASCO V-670 spectrophotometer. Emission spectra were recorded using a JASCO FP-8300 fluorescence spectrophotometer. TEM images were taken with a JEM 2100 FEG (JEOL) transmission electron microscope at an accelerating voltage of 200 kV. High resolution XPS (ULVAC-PHI XPS, PHI Quantera SXM) was used to determine atomic compositions.

Examination of the Peroxidase-like Activity of BSA-AuNCs

The peroxidase-like activity of BSA-AuNCs was evaluated in acetate buffer (pH 4, 0.1 M) with TMB (0.2 mM) as the peroxidase substrate in the presence of 50 mM H2O2. HRP was used as the positive control. For the added fluorescence quenchers, various concentrations of quenchers, including DA, AA, Hg2+, Cu2+, DTTC I, and NO2–, were first mixed with BSA-AuNCs (10 μL, 0.5 mM) for 10 min. Subsequently, acetate buffer, H2O2, and TMB were added. After the solution was incubated for 30 min, the color change of the resulting solution was observed with the UV–vis absorption spectrophotometer and photographs were taken. To monitor the catalytic oxidation of TMB with time, absorption values were collected at 652 nm for TMB using the scanning kinetic mode of the UV–vis spectrophotometer.

Detection of the Peroxidase-like Activity of BSA-AuNCs under Light Irradiation

To examine the peroxidase-like activity of BSA-AuNCs enhanced under light irradiation, experiments were carried out using 5 μM BSA-AuNCs solution in a reaction volume of 1 mL acetate buffer (pH 4, 0.1 M) with 0.2 mM TMB and 50 mM H2O2. The above solution was irradiated with a 300 W Xe lamp equipped with a cutoff filter (λ ≥ 400 nm) to provide visible light.

Acknowledgments

We thank the Ministry of Science and Technology of Taiwan (MOST-108-2113-M-030-009) for providing financial support for this research. Special thanks go to Swee Lan Cheah for her assistance on XPS measurements.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02956.

  • Absorption and emission spectra and TEM images for characterization of BSA-AuNCs; absorption spectra for the peroxidase-like activity of Cu2+ in the presence of Histidine; peroxidase-like activity of Cu2+ with HSA and the possibility of application as a sensing platform; examination of the fluorescence quenching of BSA-AuNCs by I– ions; effect of visible light irradiation on the peroxidase-like activity of BSA-AuNCs with quenchers; fluorescence quenching of BSA-AuNCs by Cu2+ at pH 4; TEM images and the size distribution of PVP-AuNPs; deconvoluted XPS spectra of PVP-AuNPs and BSA-AuNCs without and with the addition of Hg2+; effect of NaBH4 on the peroxidase-like activity of BSA-AuNCs; and peroxidase-like activities of PVP-AuNPs in the presence of DTTC I, KI, and NaI, respectively (PDF)

Author Present Address

† Department of Chemistry, National Tsing-Hua University, Hsinchu City 300, Taiwan.

Author Contributions

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

The authors declare no competing financial interest.

Supplementary Material

ao0c02956_si_001.pdf (602KB, pdf)

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