Synthesis of fluorescent α-chymotrypsin A-functionalized gold nanoclusters and their application to blot-based technology for Hg²⁺ detection

Abstract

Blot-based technology is widely used in biomedical research, serving as a remarkably efficient platform for biomolecule recognition and detection. In this report, highly fluorescent gold nanoclusters have been synthesized under mild conditions by using a proteolytic enzyme, α-chymotrypsin A (CTRA), as both the stabilizing and reducing agents. The synthesized AuNCs@CTRA was characterized by various techniques including UV-vis absorption, fluorescence, X-ray photoelectron spectroscopy and TEM. The fluorescent AuNCs@CTRA is fairly stable and responsive to mercury ions with high selectivity and sensitivity. These protein capped nanoclusters were electrophoresed on an SDS-PAGE gel and transferred to a cellulose membrane. Mercury ions can specifically quench the red fluorescent AuNCs@CTRA band and selectively stop the green band formation on the membrane through inhibition of the peroxidase mimic activity of AuNCs@CTRA toward the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate in a concentration dependent manner. Therefore, using a blot-technology based system, we demonstrated the operation of the AuNCs@CTRA–cellulose hybrid material for mercury ion visual sensing that can be dually read out under UV light (fluorometric) and the naked eye (colorimetric). This approach also has potential for use in other blot-technology based applications.

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Introduction

Gold nanoclusters (AuNCs) are sub-nanometer core sized clusters, typically consisting of a few to a hundred gold atoms. The ultra-small size of AuNCs, which is between gold atoms and nanoparticles, approaches the Fermi-wavelength of conduction electrons, resulting in molecule-like properties including discrete electronic states and size-dependent fluorescence emission.^1,2 AuNCs have gained considerable attention as novel fluorescence markers and show great promise for applications in different fields such as sensing, imaging, photo-voltaic and luminescence devices.^3–5

Recently, biomolecules have been reported to be capable of directing the synthesis of fluorescent metal nanoclusters.^6–8 For instance DNA and peptides have been employed to stabilize and template gold NCs in a “green” route.^9–12 Xie and coworkers synthesized protein-protected AuNCs using bovine serum albumin (BSA) as the stabilizing and reducing agent.¹³ AuNCs@BSA consist of 25 gold atoms (Au₂₅) with a red emission at λ_em = 640 nm. Recently, pepsin was applied by Hideya et al. to generate nanoclusters with different sizes by controlling the pH of the reaction.⁶ Pradeep et al. showed the capability of lactoferrin in the synthesis of FRET exhibiting AuNCs.¹⁴ Compared to other fluorescence materials, these protein-protected nanoclusters are non-toxic, stable, and emission wavelength-tunable. Moreover, the presence of abundant chemically reactive groups in the metal clusters–protein hybrid materials enables further functionalization of AuNCs through simple manipulations.^12,15,16 Protein protected AuNCs thus are particularly promising for a variety of applications in biomedical research. The utilization of this novel AuNCs@proteins for biosensing, cell imaging and cancer therapy has been demonstrated recently.^16–18

In addition to these biotechnologies, a widely used method in molecular biology is blot-based technology, for instance Southern, northern and western blotting. We reasoned that AuNCs@proteins have potential for use in these blot-base applications based on their special features such as fluorescent emission, protein-capping and highly sensitive to the environmental changes. To test this possibility, we screened a number of proteins such as mucin, cytochrome c, collagen and several enzymes involved in bacterial fatty acid synthesis, looking for targets can relatively tolerate harsh pH changes during the synthesis of AuNCs. To this end, a candidate called α-chymotrypsin A (CTRA) showed promising properties in that the synthesis time of AuNCs@CTRA was much shorter than that of AuNCs@BSA and other protein capped and the structure of the protein was largely maintained during the synthesis (see below).

CTRA is a 25 kDa proteolytic protein with an isoelectric point of 9.1. Compared with BSA that contains 35 thiol-containing cysteine residues in the polypeptide,¹³ CTRA has 12 cysteine residues, which suggests that CTRA may exhibit different properties as a template in the synthesis of gold nanoclusters. CTRA preferentially catalyzes the hydrolysis of peptide bonds involving L-isomers of tyrosine, phenylalanine, and tryptophan. It also readily acts upon amides and esters of susceptible amino acids. CTRA is widely used in trauma or surgery, wound healing rhinitis, hemorrhage, otitis media, as an anti-inflammatory agent and for local edema prevention. Furthermore, CTRA is a good model to study the non-native protein aggregation that is critical in amyloidosis disorders and for the development of biopharmaceuticals.^19–21 Therefore, it is also very important to develop new probes for the study of CTRA related biological questions. In this study, the synthesized AuNCs@CTRA are highly fluorescent, stable, and proved to be an efficient Hg²⁺ probe. These properties triggered us to build a new platform for heavy metal ion detection by taking advantage of blotting technology.

Heavy metal ions such as mercury ions can damage the central nervous system of mammals and are a notorious source of heavy metal pollution. Among the various applications of gold nanoclusters,^22,23 the detection of heavy metal ions has been reported, and a number of sensors to detect Hg²⁺ ions have been developed.^24–29 However, most of these methods suffer from certain limitations, including low sensitivity, poor selectivity, time-consuming and requiring expensive equipment. In this work, we report the use of CTRA to template the growth of fluorescent AuNCs using a simple, mild and rapid approach followed by the preparation of an AuNCs@CTRA–cellulose membrane hybrid material. AuNCs@CTRA band on the membrane turned red upon UV irradiation and green when it was incubated 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate. The red fluorescent band can be quenched specifically and selectively by mercury ions. In parallel, the green band formation was ceased efficiently by mercury ions through inhibition of peroxidase mimic activity of AuNCs@CTRA toward ABTS substrate. Using a blot-technology based system, we demonstrated that AuNCs@CTRA–cellulose hybrid material can serve as mercury ion visual sensor that can be dually readout under UV light (fluorometric) and naked eye (colorimetric). The proposed method is efficient, convenient and holds several advantages compared to other recently developed AuNCs-based mercury ion probe (Table S1†). This approach also has potential for use in other blot-technology based applications in environmental science or molecular biology.

Experimental section

Materials

Chloroauric acid (HAuCl₄·4H₂O), sodium hydroxide (NaOH), and other chemicals, including CaCO₃, Co(NO₃)₂, HgCl₂, Mg(NO₃)₂, Ni(NO₃)₂, Pb(NO₃)₂, Zn(NO₃)₂, Mn(NO₃)₂, Cu(NO₃)₂, FeCl₃ and FeCl₂, were purchased from Beijing Reagent Company. CTRA, BSA, Ponceau S and ABTS were purchased from Kayo (Shanghai, China) and Genview (Beijing, China), respectively. The enzymatic activity measurement of CTRA@AuNCs was carried out as follows: the substrate BSA (0.5 mg ml⁻¹) was mixed with different concentrations of CTRA@AuNCs (30, 7, and 3 mg ml⁻¹), and the mixture was incubated at 37 °C for 3 h and then subjected to SDS-PAGE. The glassware was cleaned in aqua regia and further ultrasonicated in ethanol for 10 min before use. Double distilled water (dH₂O) (18.25 mΩ) was used for all the experiments.

Instruments

UV-visible spectra were obtained using a UV-3600 spectrophotometer (SHIMADZU, Japan). X-ray photoelectron spectroscopy (XPS) data were collected on a Kratos AXIS X-ray photoelectrons spectrometer. Fluorescence spectra were recorded using a Fluorescence Max-P fluorometer (HORIBA Jobin Yvon Inc., USA) with a constant temperature control at 25 °C for steady state data. The Fourier transform infrared (FTIR) spectra were measured using a Bruker VERTEX 66V. The circular dichroism (CD) spectra were obtained using a Biologic PMS 450. Transmission electron microscopy (TEM) images were examined on a JEOL 2010 LaB6 TEM (TECNAI G2, the Netherlands) at an acceleration voltage of 200 kV.

Synthesis of AuNCs@CTRA

The procedure for the synthesis of AuNCs@CTRA was modified from Xie et al.¹³ (Scheme 1). Briefly, 250 μl of 750 μM CTRA aqueous solutions and 250 μl of 5 mM HAuCl₄ aqueous solution were mixed. Notably, the color of the solution changed from yellow to brown after stirring for 2 minutes. 29 μl of 1 M NaOH was then added into the mixture and incubated for 1–6 h at 37 °C. The above mixture was dialyzed against dH₂O until the residual NaOH and other salt ions were removed completely and then lyophilized for further use.

Scheme 1 Schematic illustration of CTRA templated synthesis of fluorescent AuNCs and their application to blot technology based mercury ion detection.

Production of AuNCs@CTRA–cellulose hybrid

CTRA and AuNCs@CTRA were heated at 95 °C for 10 min and electrophoresed on a 12% SDS-PAGE gel for approximately 2 h (100 voltages) in the presence of standard electrophoresis running buffer. The SDS-PAGE gel containing CTRA and AuNCs@CTRA was carefully packed with a cellulose membrane and 3 mm filter papers and put into a standard western-blot cassette containing electro-transfer buffer (Scheme 1 and Fig. 4a). AuNCs@CTRA were then transferred to a cellulose membrane after 90 minutes (100 voltages). The cellulose membrane after this transferring process was washed with dH₂O before signal detection and then expose to UV light for image collection or ready for other application. Notably, AuNCs@CTRA–cellulose hybrid is very stable for at least one month in 4 °C refrigerator, which make this material very convenient to be used.

Blot-based metal ion detection using a UV light

The CTRA and AuNCs@CTRA containing SDS-PAGE gel and blot (cellulose membrane) can be stained with Coomassie blue or analyzed under UV light. Metal ions with certain concentrations (10 μM, 1 μM, 10 nM, 8 nM, 5 nM and 1 nM) were incubated with AuNCs@CTRA containing membrane. Images were then collected under UV irradiation. All of the experiments were repeated at least three times.

Blot-based metal ion detection by naked eye

For ABTS-blot-based peroxidase artificial enzyme activity assay, AuNCs@CTRA containing membrane was incubated with 150 μM ABTS and 0.3% H₂O₂ in 200 mM NaAc buffer (pH 4), at 40 °C for 15 min. For metal ion detection, metal ions with certain concentrations (10 μM, 1 μM, 10 nM, 8 nM, 5 nM and 1 nM) were incubated with AuNCs@CTRA hybrid membrane. The resulting membrane was then incubated with ABTS and 0.3% H₂O₂ in 200 mM NaAc buffer (pH 4) at 40 °C for 15 min. All of the experiments were repeated at least three times.

Results and discussion

Spectroscopic characterization of AuNCs@CTRA

AuNCs@CTRA showed a red fluorescence emission as visualized on fluorescence emission spectra (Fig. 1a). To monitor the whole synthesis process and to optimize the reaction time, the fluorescence spectra of the reaction mixtures were recorded at fixed intervals (Fig. S1†). Obvious fluorescence signals from the AuNCs generated could be observed after 1 h reaction, and increased gradually thereafter. Specifically, an emission band centered near 638 nm was visualized and gradually enhanced, confirming that AuNCs@CTRA were forming and growing during the process. The fluorescence intensity of AuNCs@CTRA achieved a maximum after 6 h reaction, thus this time was chosen for the AuNCs synthesis. It is important to note that, the optimum time for the synthesis of CTRA templated gold nanoclusters is much short than the 12 h reported for the popular BSA templated gold nanocluster synthesis.¹³

Fig. 1 Fluorescence, UV-vis spectra characterization of AuNCs@CTRA. (a) Fluorescence excitation (the red line, excitation at 375 nm) and emission spectra (the black line, emission at 615 nm) of AuNCs@CTRA. (b) UV-vis spectra of AuNCs@CTRA, the red and black lines represent AuNCs@CTRA and pure CTRA, respectively.

The CTRA protein powder appeared white under visible light and emitted blue light under UV excitation (300 nm) (Fig. 2a), whereas the AuNCs@CTRA both as a lyophilized powder (Fig. 3b) and in aqueous solution (Fig. 2c) were brown under visible light and showed red fluorescence under UV light (300 nm).

Fig. 2 Images of AuNCs@CTRA recorded by digital camera under visible and UV light. (a) CTRA protein powders under visible and UV light (b) lyophilized AuNCs@CTRA under visible and UV light and (c) AuNCs@CTRA solution under visible and UV light.

Fig. 3 AuNCs@CTRA response to mercury ions in aqueous solution with high selectivity and sensitivity. (a) Fluorescence emission spectra of AuNCs@CTRA (0.1 mg ml⁻¹) with the concentrations of Hg²⁺ increasing from 0 to 10 μM (top to bottom). (b) Relative fluorescence intensity of AuNCs@CTRA (F/F₀, F and F₀ refer to the fluorescence intensities in the presence and absence of Hg²⁺) versus the logarithm of the Hg²⁺ concentrations. (c) Fluorescence response of AuNCs@CTRA (F/F₀) in the presence of different metal ions (the metal ions concentration at 10 μM) practical application of AuNCs@CTRA as a Hg²⁺ ions selective fluorescence probe, we carried out a competitive experiment by mixing Hg²⁺ ions (10.0 μM) with other metal ions (10.0 μM) (Fig. S8†). The results showed negligible influences of these metal ions on the fluorescence response of AuNCs towards Hg²⁺ ions.

Notably, the quantum yield of AuNCs@CTRA in water is 4.6%, calculated using rhodamine 6G as the reference. As shown in Fig. 1b, the absorption spectrum of CTRA in aqueous solution showed a characteristic peak at 280 nm, which was correlated to the absorption of aromatic amino acids in the proteins. The UV spectrum of AuNCs@CTRA showed a shoulder in this region with a continuous rise from 600 nm to 400 nm, which resembled the spectrum of AuNCs@BSA.

XPS and TEM characterization of AuNCs@CTRA

XPS measurements were carried out to detect the valence states of gold within the nanocluster holding proteins. The binding energy of Au 4f_7/2 and Au 4f_5/2 for AuNCs@CTRA was 84.1 eV and 87.7 eV, respectively (Fig. S2a†). It should be noted that the binding energy of Au 4f_7/2 in Au(0) XPS spectra correlates to 84.0 eV and the binding energy of Au(I) correlates to 85.0 eV.^30–32 The Au 4f_7/2 spectrum of AuNCs@CTRA can be further deconvoluted into two distinct components (dot curves) centered at 84.0 and 85.0 eV, which are assigned to Au(0) and Au(I), respectively. The integrated area of these two bands illustrates that the majority of the gold within the nanoclusters is Au(0) in the core complemented with a minor amount of Au(I) at the core surface, which might stabilize the AuNCs. The Au 4f XPS spectrum of the reaction intermediate suggests the Au³⁺ ions were reduced to Au(0) atoms successfully by CTRA by using the protocol we applied. To visualize the gold nanoclusters synthesized using CTRA as a template, the AuNCs@CTRA samples were investigated using TEM microscopy. A representative TEM micrograph of the AuNCs@CTRA nanoclusters showed uniformly distributed particles that were essentially spherical. The average size of the nanoclusters was approximately 2 nm (Fig. S2b† and data not shown), which fits well with the observations from UV-vis absorption and fluorescence spectra.

The stability and fluorescence decay of AuNCs@CTRA

For the biological applications of metal nanoclusters, it is always important to evaluate their stability before use. Interestingly, the AuNCs@CTRA were fairly stable and was well suitable as a fluorescence probe even several months after synthesis, as tracked by fluorescence measurement (data not shown). Moreover, we carefully examined the colloidal stability in solution over a wide pH range. The steady fluorescence intensity of AuNCs@CTRA was basically constant under various pH conditions within the range of 6–13 (Fig. S3†). The properties of fluorescence decay of the AuNCs@CTRA were further monitored under neutral pH conditions. The decay curve of AuNCs@CTRA in HEPES buffer (pH 7.5) showed three components, at 47.9 ns (1.1%), 422.4 ns (8.9%), and 1950.79 ns (90%) (Fig. S4†). This indicated that the major fluorescent constituent within the materials has a similar or even relatively longer lifetime than previously reported for other AuNCs capped by proteins and thiolates,^33,34 which might be related to the properties of Au(I)–thiol complexes that exist on the AuNC surfaces.

Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy

As a protein–gold nanocluster hybrid material, we were interested to know whether or not the protein within the material was structurally and/or functionally retained as compared to the native enzyme, which is also critical concern for blot-based technology. FT-IR and CD spectroscopy were thus employed to reveal the structural features of the CTRA part. In the FT-IR spectra, the bending and stretching vibrations of the peptide backbone that characterize the secondary structure and the position of these bands in AuNCs@CTRA remained largely the same (Fig. S5†), which indicates that the fingers of the capping molecules of the nanoclusters are maintained during the synthesis. To examine further the potential conformational changes of CTRA that might occur during the process of the nanoclusters synthesis, CD spectra were carried out to monitor the secondary structure of the protein–gold nanocluster hybrid. The intensity of the peak at 205 nm of the AuNCs@CTRA was decreased and the CD curve also changed slightly in the shoulder regions (red curve, Fig. S6†) as compared to the pure CTRA enzyme. This indicates that a slight conformational change occurred when CTRA capped on the AuNCs surface. The loss of CTRA enzymatic activity in the material might attributed to the damage of catalytic site of the enzyme during the synthesis process (Fig. S7†). We are currently working on this aspect through manipulation of the synthesis protocol, with the goal of preparing a nanocluster hybrid system containing active enzymes.

Metal ion detection in aqueous solution

Metal ion detection was among one of the promising applications of fluorescent AuNCs. Recent studies have proposed that mercury ions can quench the fluorescence emission of the protein protected AuNCs due to the metallophilic interaction with the d¹⁰–d¹⁰ orbitals.^33,34 To test the capability of AuNCs@CTRA in this respect, the fluorescence emission spectra of the materials were recorded in the presence of Hg²⁺. As shown in Fig. 3a, the fluorescence intensity of our AuNCs@CTRA decreased significantly with increasing concentrations of Hg²⁺. To investigate the detection limit of the nanoclusters toward the Hg²⁺ ions, 0.1 mg ml⁻¹ AuNCs@CTRA was used to measure the sensitivity. Our results showed that AuNCs@CTRA respond to the mercury ions linearly within the range of 10⁻⁹ to 2 × 10⁻⁶ M (Fig. 3b), and the detection limit approached 1 nM.

Many transition metal ions, e.g., Ca²⁺, Zn²⁺, Fe³⁺, Cu²⁺, Mn²⁺, Co²⁺ and Ni²⁺, are essential in living organisms. Moreover, a number of heavy metal ions, e.g., Ag⁺, Pb²⁺, and Cd²⁺, were notorious in pollution investigation. Therefore, the responses of these metal ions were also examined in this study to evaluate the specificity of our system. Notably, the fluorescence of the AuNCs@CTRA was quenched only by Hg²⁺, while no such effect was observed for the other metal ions tested (Fig. 3c), which suggests a high selectivity of the CTRA stabilized nanoclusters for mercury ion detection. To further evaluate the practical application of AuNCs@CTRA as a Hg²⁺ ions selective fluorescence probe, we carried out a competitive experiment by mixing Hg²⁺ ions (10.0 μM) with other metal ions (10.0 μM) (Fig. S8†). The results showed negligible influences of these metal ions on the fluorescence response of AuNCs towards Hg²⁺ ions.

SDS-PAGE and AuNCs@CTRA–cellulose hybrid

Blot-based technology such as southern, northern and western blotting is widely used in biomedical research. The basis of this technique is transfer biomolecules such as proteins, DNAs, or RNAs to a thin layer such as cellulose or PVDF membrane to form a stable hybrid material (Scheme 1 and Fig. 4a). The hybrid thin layer is served as a platform for signal detection. The properties characterized above indicate that AuNCs@CTRA can be a candidate to be used in blot-based technology for metal ions detection. To test this possibility, CTRA and AuNCs@CTRA were electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The position of the protein bands on the gel were visualized by Coomassie blue staining (Fig. 4b). As expected, the AuNCs@CTRA showed a red-emitting band when exposed to UV light whereas CTRA by itself was not visible (Fig. 4c). Noteworthily, CTRA–AuNCs complex underwent automatic cross-linking between certain amino acid groups within CRTA proteins under the alkali synthesis condition, and thus forming a microgel-like structure. This explained that a shift of band position happened on the SDS-PAGE gel between CRTA and AuNCs@CTRA (Fig. 4b–e). The CRTA and CRTA–AuNCs complex were transferred onto a cellulose membrane using a western-blot apparatus (Scheme 1, Fig. 4a, d and e). The protein bands on the membrane can be visualized by Ponceau S staining (Fig. 4d). Similar to CRTA and CRTA–AuNCs containing SDS-PAGE gel, the band of AuNCs@CTRA on the cellulose membrane was red-emitting upon UV irradiation (Fig. 4e).

Fig. 4 SDS-PAGE and AuNCs@CTRA blotting. (a) Scheme of a transferring set for making AuNCs@CTRA–cellulose hybrid. (b) SDS-PAGE gel of CTRA and AuNCs@CTRA stained with Coomassie blue. (c) The CTRA and AuNCs@CTRA loaded SDS-PAGE gel under UV exposure. (d) CTRA and AuNCs@CTRA–cellulose film stained with Ponceau S solution. (e) CTRA and AuNCs@CTRA–cellulose film under UV exposure. Arrow and arrow head point to the CTRA before and after AuNCs synthesis, respectively.

Mercury ion detection using AuNCs@CTRA–cellulose as fluorometric sensor

The fluorescent signals of AuNCs@CTRA are positively correlated with the amount of AuNCs@CTRA loaded onto the SDS-PAGE gel, with a detection limit of approximately 10 μg (referring to the quantity of CTRA) (Fig. 5a and b). This suggests that there is room to manipulate the system via changing the amount of materials loaded to gels. To test whether or not this AuNCs@CTRA loaded thin layer can serve as a new type of sensor with special reference to mercury ion detection, the membranes were incubated with different metal ions at fixed concentrations between 1 nM and 10 μM. As shown in Fig. 5c–e. Hg²⁺ ions efficiently quenched nearly completely the red fluorescent band on the film at 10 nM, whereas other metal ions such as Mn²⁺, Mg²⁺, Fe³⁺, Fe²⁺, Zn²⁺ and Ca²⁺ did not have any visible impact on the red fluorescent signals at 10 nM to 10 μM, indicating the high selectivity of this AuNCs–blot–UV-based method for mercury ion detection. Moreover, impairment towards red signal can be observed when concentration of Hg²⁺ ions reaches 8 nM (Fig. 5f), suggesting a limit of detection (LOD) less than 10 nM. These data clearly hinted that the AuNCs–blot–UV system could effectively probe mercury ions by using a UV light, serving as a highly selective and promising sensor for metal ion detection.

Fig. 5 AuNCs@CTRA on cellulose membrane response to mercury ions with high selectivity and sensitivity as shown by the red fluorescent signal changes detected under UV light. (a) Scheme shows the working mechanism of the fluorometric probe. (b) The fluorescent signals of AuNCs@CTRA are positively correlated with the amount of AuNCs@CTRA loaded onto the SDS-PAGE gel, with a detection limit of 10 μg (referring to the quantity of CTRA). (c) AuNCs@CTRA on the film under UV exposure in the presence of dH₂O (upper panel) or metal ions (lower panel) at 10 nM (c), 1 μM (d) and 10 μM (e), respectively. At the given concentrations, mercury ions quenched the red fluorescent signal whereas other metal ions do not. (f) AuNCs@CTRA on the film emitted fluorescent bands under UV exposure in the presence of dH₂O (upper panel) or quenched by mercury ions (lower panel) at 1–10 nM. The detection limit of this UV-based sensor for mercury ion detection was determined to be 8 nM.

Mercury ion detection using AuNCs@CTRA–cellulose as colorimetric sensor

Recent advance indicated that certain nanomaterial for instance AuNCs@BSA contain intrinsic enzyme mimic activities such as peroxidase activity.^35–37 Our data showed that AuNCs@CTRA also exhibit peroxidase mimic activity toward 3,3′,5,5′-tetramethylbenzidine (TMB). However, this far most of the substrates and products used in mimic enzymatic measurements were soluble. This limit the use of nanomaterial in blot-based technology especially those requiring product precipitated on membrane. Several molecules frequently used in western blotting experiments (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP), 4-chloro-1-naphthol (4-CN), 3,3-diaminobenzidine tetrahydrochloride (DAB), enhanced chemiluminescence (ECL) substrate, nitro-blue tetrazolium chloride (NBT), and TMB) were carefully examined using AuNCs@CTRA–cellulose hybrid membrane. Interestingly, AuNCs@CTRA can convert the colorless soluble ABTS into a green insoluble form of product that precipitates next to the nanoclusters and thus stains the thin layer (Fig. 6a and b), whereas all the other substrates test so far failed. This phenomenon can be explained by the unsuitable working pH (the optimal pH for the peroxidase mimic activity of AuNCs is approximately 4) and solubility of these systems for the blot-based application. Strikingly, with the addition of mercury ions, the enzyme mimic activity of AuNCs@CTRA decreased dramatically, which is likely due to the inhibition of Hg²⁺ towards the peroxidase mimic function of the gold nanoclusters. Therefore, this ABTS based AuNCs@CTRA–cellulose system can serve as the colorimetric sensor for the detection of mercury ions.

Fig. 6 AuNCs@CTRA on cellulose membrane response to mercury ions with high selectivity and sensitivity as shown by the visual green signal changes. (a) Scheme shows the working mechanism of the colorimetric probe. AuNCs@CTRA converts ABTS into insoluble green product. (b) The green signal is positively correlated with the amount of AuNCs@CTRA loaded onto the SDS-PAGE gel, with a detection limit of 10 μg (referring to the quantity of CTRA). (c) AuNCs@CTRA on the film under ABTS based enzyme assay condition in the presence of dH₂O (upper panel) or metal ions (lower panel) at 10 nM (c), 100 nM (d) and 10 μM (e), respectively. At the given concentration, mercury ions inhibit the green color development catalyzed by AuNCs@CTRA whereas other metal ions do not. (f) AuNCs@CTRA catalyze the green band formation on the film in the presence of dH₂O (upper panel) or inhibited by mercury ions (lower panel) at 1–10 nM. The detection limit of this colorimetric sensor for mercury ion detection was determined to be 8 nM.

The green signal is positively correlated with the amount of AuNCs@CTRA loaded onto the SDS-PAGE gel, with a detection limit approximately 10 μg (referring to the quantity of CTRA) (Fig. 6b). Similar to the UV-based system, AuNCs@CTRA–cellulose membrane was incubated with different metal ions at fixed concentrations. As shown in Fig. 6c–e the film containing 10 nM Hg²⁺ ions was nearly colorless, while other metal ions such as Mn²⁺, Mg²⁺, Fe³⁺, Fe²⁺, Zn²⁺ and Ca²⁺ at 10 nM to 10 μM exhibited a deep green color, indicating the high selectivity of this visual method for mercury ion detection. Importantly, the remarkable selectivity toward Hg²⁺ over other metal ions could be observed by naked eye. Moreover, there is impairment of green signal when concentration of Hg²⁺ ions approaches 8 nM (Fig. 6f), suggesting a low LOD of this visual system. Our results demonstrated that combining with ABTS–H₂O₂ as substrates, AuNCs@CTRA–cellulose membrane could effectively recognize mercury ions and signals readily detectable with naked eye. Therefore, this platform can serve as a promising colorimetric probe for metal ion sensing. Notably, the LOD for mercury ion detection using this visual system can be as low as 8 nM, that is comparable to the AuNCs–blot–UV fluorometric mercury ion probe mentioned above.

In summary, the LOD of the AuNCs@CTRA–cellulose membrane based Hg²⁺ sensor was 8 nM, which meets the analytical standard drinking water of the U.S. EPA (10 nM).³⁸ In addition to its high selectivity and sensitivity, this thin layer sensor was very convenient to use and the signal is readily detectable by using a UV light or simply the naked eyes. Moreover, no expensive equipment is required for the test.

Conclusions

α-chymotrypsin A (CTRA), as an important enzyme in medical research, was used to direct the synthesis of gold nanoclusters in a mild condition. AuNCs@CTRA were characterized using UV-vis, fluorescence, XPS spectroscopy and TEM. The AuNCs synthesized using “green method” was transferred to a cellulose membrane by taking advantage of blotting technology. The AuNCs@CTRA–cellulose membrane can serve as an efficient sensor for mercury ion detection with high selectivity and sensitivity. The signals from this dual role system can be easily readout under a UV light (fluorometric) or visually detected by naked eye (colorimetric). Based on these results, we propose that this system could act as an alternative analytic tool for Hg²⁺ detection in life sciences and pollution research.

Acknowledgements

This work was supported by the Start-up funds from the State Key Laboratory of Supramolecular Structure and Materials at Jilin University, the National Natural Science Foundation of China (NSFC) (no. 21372097 and no. 21003061), Open Fund of the National Laboratory of Protein and Plant Gene Research at Peking University, and a CIMO Grant from Finland.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Reaction time of AuNCs@CTRA synthesis, pH stability, Fluorescence decay, FT-IR, CD spectra, CTRA activity measurement and the inference of other metal ions toward AuNCs@CTRA sensor. See DOI: 10.1039/c4ra05686h

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