An Au@NH₂-MIL-125(Ti)-based multifunctional platform for colorimetric detections of biomolecules and Hg²⁺

Abstract

In this report, a novel nanocomposite of highly dispersed Au nanoparticles deposited on NH₂-MIL-125(Ti) was designed and proposed as a peroxidase-like mimic. Compared with individual Au nanoparticles and NH₂-MIL-125(Ti), the peroxidase-like nanozyme exhibits enhanced peroxidase-like activity. Moreover, kinetic analysis reveals that the as-prepared nanozyme has lower K_m values and stronger affinity towards both substrates than the natural horseradish peroxidase (HRP). The detection limits of the multifunctional platform for H₂O₂, cysteine and Hg²⁺ are down to 0.24 μM, 0.14 μM and 100 nM, respectively. This work provides rapid and sensitive colorimetric detection strategies for small biomolecules and Hg²⁺, which have great potential for application in biosensors and biotechnology.

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1. Introduction

H₂O₂ and cysteine play crucial roles in biological systems and their abnormal levels have been proved to be risk factors of diverse diseases. Moreover, even a low content of mercury(II) ions (Hg²⁺) can lead to damage in the brain, kidney, nerve and endocrine system.^1–3 Thus, sensitive detection of these target molecules is highly desirable. Among various reported detection strategies for these target molecules and metal ions, the enzyme-based colorimetric sensing technique has been practiced as one of the most powerful means for the point-of-care detection of these species through a change in color, due to its rapid visual determination, high sensitivity and high-throughput.^4–8 However, its practical applications in colorimetric sensing have been severely blocked by the considerable drawbacks of natural enzymes, such as high-cost, low stability and sensitivity to harsh environments. To overcome these drawbacks, nanozymes, nanoparticle-based enzyme mimics, have been investigated intensively in the past decade in virtue of their various intriguing advantages such as high-stability, low-cost and adjustable-activity.^8–11 To date, various nanomaterials including Fe₃O₄,¹² nanoceria,^13,14 graphene oxide,¹⁵ carbon nanodots,¹⁶ metal metallic nanoparticles,^17–19etc. have been explored to mimic natural enzymes. Among the various developed nanomaterial-based enzyme mimics, metallic Au nanoparticles have been extensively recognized as peroxidase or oxidase-like biomimetics.^18–20 However, their practical applications have been inevitably blocked by intrinsic drawbacks such as easy aggregation and limited catalytic efficiency.

To address these issues, various strategies have been developed to improve the catalytic properties of metallic Au-based nanozymes. Among the developed strategies, combing Au with other support materials, such as graphene and metal organic frameworks, is promising and attractive, because the resulting composites may exhibit higher activities owing to the synergistic effect between the single components.^21–23 Due to the intrinsic advantages of metal organic frameworks, several Au/MOF composites have been fabricated and applied in catalysis and sensing.^22–28 For example, Liu et al. reported the detection of DNA using an Au@Fe-MIL-88 hybrid as a peroxidase-like nanozyme, and the nanohybrid exhibited enhanced activity compared to pure Fe-MIL-88 and Au NPs.²³ Hu et al. developed a highly sensitive detection strategy for p-phenylenediamine using Au/MIL-101 as the SERS substrate.²⁵ These studies demonstrated that Au/MOF composites have great potential for application in biomedical analysis and environmental monitoring as mimic enzymes. However, among the various reported Au@MOF composites, only Au@Fe-MIL-88 and Au/MIL-101 have been applied as nanozymes. Moreover, only Au@Fe-MIL-88 was used as a nanozyme for colorimetric sensing (DNA).

NH₂-MIL-125(Ti), a porous amino-functionalized titanium-based MOF, has been proved to be an efficient and stable support for metallic nanoparticles (Pd, Pt, and Au) and the resulting composites exhibited excellent heterogeneous catalytic performance in Suzuki cross coupling (Pd),²⁹ electrocatalytic oxidation of hydrazine (Au)²⁶ and photocatalytic alcohol oxidation (Pt and Au).³⁰ However, none of the M@NH₂-MIL-125 composites have been explored as nanozymes and applied in the colorimetric sensing of biomolecules and metal ions. Taken together, to the best of our knowledge, neither the enzyme mimic characteristic nor the application in colorimetric assays of the Au@NH₂-MIL-125(Ti) composite has been explored.

Inspired by the above considerations, as depicted in Scheme 1, a composite was prepared by depositing Au nanoparticles on NH₂-MIL-125(Ti) and its peroxidase-like characteristic and enzymatic kinetics were firstly investigated in detail. Based on the excellent peroxidase-like activity and inhibition effect of cysteine, a multifunctional platform for rapid, convenient and sensitive colorimetric detection of H₂O₂, cysteine and Hg²⁺ with very low detection limits has been developed.

Scheme 1 Schematic illustration for the preparation of Au@NH₂-MIL-125(Ti) nanocomposite and colorimetric detections of H₂O₂, cysteine and Hg²⁺.

2. Experimental

2.1. Preparation of NH₂-MIL-125(Ti)

NH₂-MIL-125(Ti) was synthesized by a conventional solvothermal procedure similar to reported methods.³¹ Generally, 1.63 g of 2-aminoterephthalate (9 mmol) was first dissolved in a dehydrated mixed solution containing 27 ml of DMF and 3 ml of MeOH in a 50 ml Teflon-lined autoclave. Consequently, 0.72 ml of tetrabutyl titanate (2.25 mmol) was dropped into the mixture under sonication in order to obtain a uniform solution. Afterward, the autoclave was sealed and heated to 150 °C in an oven for 48 h. After cooling down, the powder was separated by centrifugation. After washing with DMF and MeOH, the powder was dried at 60 °C for 6 h in a vacuum oven.

2.2. Loading of Au nanoparticles into NH₂-MIL-125(Ti)

Typically, 100 mg of NH₂-MIL-125(Ti) was first dispersed in 20 ml of ultrapure water and then 1.00 ml of HAuCl₄ (1%) aqueous solution was dropped into the mixture. After 5 min of sonication, 6 mg of sodium borohydride was added to reduce the gold ions into metallic nanoparticles. After sonicating for 20 min, the solution was centrifuged, and the solid was washed several times with methanol.

2.3. Characterization

The crystal structures of the materials were examined by X-ray powder diffraction (XRD) in the 2θ range from 5 to 80 degree using a Bruker D4 Endeavour equipped with Cu Kα radiation (λ = 0.15405 nm). The structure and morphology of the materials were analyzed by scanning electronic microscopy (SEM, Hitachi S-5500). X-ray photoelectron spectroscopy (XPS) measurement was carried out using a PHI ESCA-5000C electron spectrometer. The Au contents in NH₂-MIL-125 samples were tested using an inductively coupled plasma mass spectrometer (ICP-MS, Thermo NexION 1000).

2.4. Peroxidase-like catalytic activities of Au@NH₂-MIL-125(Ti)

The oxidation of a color substrate 3,3′,5,5′-tetramethylbenzidine (TMB) with H₂O₂ was selected as a model reaction to examine the peroxidase-like activities of Au@NH₂-MIL-125(Ti) and related materials. Catalyst aqueous suspensions (1 mg ml⁻¹) were prepared in advance by dispersing materials in water under sonication for 10 min. Typically, 0.1 ml of the catalyst suspension and 0.1 ml of TMB solution (10 mM in DMSO) were mixed well with 4.3 ml of acetate buffer solution (pH = 4.0) in a quartz cell. Finally, 0.5 ml of H₂O₂ (50 mM) was injected into the above mixture to start the reaction. The peroxidase-like activities were monitored by recording the UV-vis spectra or absorbance at 652 nm of the reaction mixture for a total of 10 min. To compare the enzymatic kinetics, we measured the steady-state kinetics for both TMB and H₂O₂ as substrates under optimum conditions. The kinetic data were obtained by changing one substrate concentration and fixing that to the other substrate. The Michaelis Menten parameters were calculated from the Lineweaver Burk plots

1/V = 1/V_max + K_m/(V_max[S])

where V and V_max represent the initial and maximum velocity, respectively, [S] is the substrate concentration, and K_m is the Michaelis constant.

2.5. Radical trapping experiments

Typically, scavengers (50 mM), such as terephthalic acid (TA) and isopropanol (TPA) as ˙OH scavengers, and benzoquinone (BQ) as an ˙O₂⁻ scavenger, were added into the H₂O₂  +  TMB + Au@NH₂-MIL-125(Ti) system while keeping other conditions fixed.

2.6. Colorimetric detection of H₂O₂ and cysteine

Typically, a colorimetric reaction was performed as follows to detect H₂O₂, 0.5 ml of H₂O₂ at varying concentrations of 0.1–80 mM was injected into a mixture containing 0.1 ml of TMB (5 mM) and 0.1 ml of the catalyst suspension (1 mg ml⁻¹) in 4.3 ml of acetate buffer solution (100 mM, pH = 4.0). The successive concentration-dependent changes of the absorbance at 652 nm of the reaction mixtures were evaluated to calculate their initial velocities and further to calculate the kinetic parameters. The colorimetric detection of cysteine is the same as the sensing of H₂O₂. Briefly, cysteine at different concentrations was mixed with 4.3 ml of acetate buffer solution (pH = 4) containing 0.1 ml of Au@NH₂-MIL-125 aqueous suspension (1 mg ml⁻¹), 0.1 ml of TMB solution (5 mM in DMSO) and 0.5 ml of H₂O₂ (50 mM). Then the ΔA at 652 nm was measured by UV-vis absorption spectroscopy. To study the selectivity for cysteine towards other biomolecules found in biosystems, Au@NH₂-MIL-125 was incubated with various amino acids, such as phenylalanine (Phe), histidine (His), glucose (Glu), tryptophan (Trp), methionine (Met), valine (Val), ascorbic acid (AA), and glutathione (GSH) instead of cysteine. And their ΔA values at 652 nm were measured to be the same as the detection of cysteine.

The feasibility of the developed assay for cysteine detection in biological samples was evaluated by spiking cysteine in fetal bovine serum, which was diluted with acetate buffer (pH = 4.0, 100 mM) at a ratio of 1 : 100. The added cysteine (4.0 μM, 7.0 μM, and 9.0 μM) in real samples was analyzed following the same procedure as described above.

2.7. Colorimetric detection of Hg²⁺

For Hg²⁺ detection, Hg²⁺ in various amounts was introduced into the acetate buffer solution (pH = 4.0) containing cysteine (60 μm) and Au@NH₂-MIL-125(Ti) (20 μg ml⁻¹). One minute later, H₂O₂ (5 mM) and TMB (0.1 mM) were added. After reacting for 10 min, the absorbances at 652 nm were recorded. Subsequently, to investigate the selectivity of Hg²⁺ towards other metal ions, the changes of the absorbance at 652 nm were determined to be the same as the detection of Hg²⁺, except for adding various other metal ions of the same concentrations instead of Hg²⁺. Moreover, in order to eliminate the interference of other ions, EDTA (1 mM) and NaCl (0.5 mM), acting as masking agents, were added together with the metal ions into the reaction mixture. The practical applicability of this colorimetric assay for the detection of Hg²⁺ in tap water was further investigated following the same procedures.

3. Results and discussion

3.1. Characterization of the synthesized materials

Scheme 1 shows a schematic illustration of the fabrication of Au@NH₂-MIL-125(Ti). Briefly, NH₂-MIL-125(Ti) was first synthesized by a solvothermal reaction. Subsequently, Au@NH₂-MIL-125(Ti) was fabricated by impregnation followed by reduction with sodium borohydride. The crystalline structures of the as-synthesized NH₂-MIL-125(Ti) and Au@NH₂-MIL-125(Ti) were analyzed first by means of the X-ray diffraction (XRD). As shown in Fig. 1a, the XRD pattern of prepared NH₂-MIL-125 shows remarkable characteristic peaks at 2θ ranging from 5 to 20°, which are in perfect agreement with those of the simulated patterns shown in Fig. 1b of NH₂-MIL-125(Ti).^31–33 Furthermore, the loading of Au NPs on NH₂-MIL-125(Ti) does not affect its crystalline structure. As shown in Fig. 1d, broad and weak peaks attributed to the Au NPs are observed, probably due to the uniform dispersion of the gold nanoparticles on NH₂-MIL-125.

Fig. 1 XRD patterns of materials. (a) As-prepared NH₂-MIL-125(Ti); (b) simulated NH₂-MIL-125(Ti); (c) simulated Au; (d) Au@NH₂-MIL-125(Ti).

The morphologies, structures and composition of the as-prepared NH₂-MIL-125(Ti) and Au@NH₂-MIL-125(Ti) were further investigated by TEM and SEM techniques. As can be observed in both SEM and TEM images (Fig. 2), the obtained NH₂-MIL-125 sample is exclusively characteristic of plate shape. The plates have an average diameter of 500 nm and a thickness of 300 nm (Fig. 2b). As shown in the TEM and SEM images, small Au nanoparticles with an average diameter less than 5 nm were observed both on the surface and in the internal pores. In addition, the incorporation of Au did not affect the shape and the structure of NH₂-MIL-125. SEM-EDX measurement was further applied to explore the existence of Au and surface composition in Au@NH₂-MIL-125(Ti). As shown in Fig. 2g, peaks assigned to both Ti and Au elements were observed in the spectra. The Au loading amount was quantitatively determined by ICP-MS, and the weight percent was calculated to be about 3%. All the results described above well demonstrated that Au nanoparticles were incorporated on and into NH₂-MIL-125(Ti) without destroying its crystalline structure and shape.

Fig. 2 TEM, SEM images and SEM-EDX spectra. (a) SEM images of NH₂-MIL-125(Ti); (b and c) Au@NH₂-MIL-125(Ti); TEM images of (d) NH₂-MIL-125(Ti) and (e and f) Au@NH₂-MIL-125(Ti); and SEM-EDX spectra (g) of Au@NH₂-MIL-125(Ti).

The surface compositions and chemical states of the NH₂-MIL-125(Ti) and Au@NH₂-MIL-125(Ti) composites were further investigated by X-ray photoelectron spectroscopy (XPS) measurements. Fig. 3a shows the survey XPS spectra of the NH₂-MIL-125(Ti) (red line) and Au@NH₂-MIL-125(Ti) (black line) samples. The results clearly reveal that the Au@NH₂-MIL-125(Ti) composite mainly consists of C, N, O, Ti and Au atoms while there are no Au atoms in the NH₂-MIL-125, which is in good agreement with their SEM-EDX results. The Ti 2p spectrum of the Au@NH₂-MIL-125(Ti) sample shows two contributions, located respectively at 458.8 and 464.5 eV, which are assigned to be the binding energy values of Ti 2p_3/2 and Ti 2p_1/2, indicating that titanium bound to oxygen remains in oxidation state IV for the titanium–oxo cluster.³³ As shown in Fig. 3c, the presence of the reduced form of Au⁰ in the Au@NH₂-MIL-125(Ti) was evidenced by the Au 4f_7/2 and Au 4f_5/2 peaks located respectively at 83.9 and 87.6 eV. Furthermore, the shift of the N 1s binding energy of Au@NH₂-MIL-125 when compared with that of pristine NH₂-MIL-125(Ti) provides evidence of an electronic interaction between the N of the amine functionality and the deposited Au nanoparticles.³³

Fig. 3 XPS spectra of NH₂-MIL-125(Ti) and Au@NH₂-MIL-125(Ti). (a) Survey; (b) Ti 2p; (c) Au 4f; and (d) N 1s.

3.2. Peroxidase-like catalytic activity

In order to explore the peroxidase-like catalytic properties of the as-prepared Au@NH₂-MIL-125(Ti), the oxidation of TMB with the aid of H₂O₂ was tested. TMB is a chromogenic substrate which would display blue color in the presence of peroxidase and H₂O₂. The concentration of the oxidized product ox-TMB can be quantitatively determined by measuring the absorbance values at 652 nm. Therefore, the peroxidase-like activities were monitored by recording the time-dependent absorbance UV-vis spectra or absorbance at 652 nm of the reaction mixture at a 1 min interval for a total of 10 min. As shown in Fig. 4a, the absorbance of the reacted solutions at 652 nm increased linearly in the time-dependent absorbance spectra, resulting from the formation of a charge transfer complex and a radical cation. And then, we conducted a series of control experiments to further explore the peroxidase-like activity of the Au@NH₂-MIL-125 hybrid. As shown in Fig. 4b, TMB and H₂O₂, TMB with Au@NH₂-MIL-125(Ti), and H₂O₂ with Au@NH₂-MIL-125(Ti) systems are colorless or light blue and display weak absorbance peaks at 652 nm, while TMB, H₂O₂ and Au@NH₂-MIL-125(Ti) system display a dark blue color and a strong absorbance peak at 652 nm, indicating that the Au@NH₂-MIL-125(Ti) nanohybrid is an efficient peroxidase-like biomimetic which can catalyze the reaction of H₂O₂ oxidizing TMB to form ox-TMB. As shown in Fig. 4c, it is worth mentioning that Au@NH₂-MIL-125(Ti) exhibits stronger peroxidase-like activity than single NH₂-MIL-125(Ti) and Au nanoparticles. Herein, we speculate that the enhancement is probably due to the synergistic effect between the single components of the nanohybrid. In order to obtain the best catalytic activity, we further investigated the influence of pH values on the catalytic activity of Au@NH₂-MIL-125(Ti) because the peroxidase-like activity of nanozymes is sensitive to the pH values.^11,34–36 As shown in Fig. 4d, like the natural enzymes and most reported peroxidase-like nanozymes, the highest catalytic activity was found at pH = 4.0. Therefore, the optimal pH value for the reaction system is 4.0.

Fig. 4 (a) Time-dependent changes of UV-vis absorption curves of TMB oxidation (0.2 mM) in the presence of H₂O₂ (5 mM) and the Au@NH₂-MIL-125(Ti) nanohybrid (20 μg ml⁻¹) in acetate buffer solution (pH = 4.0). The inset shows the plot of absorbance at 652 nm versus time; (b) UV-vis absorption curves of different reaction systems after 10 min, the TMB, H₂O₂ and catalyst concentrations are 0.2 mM, 5 mM and 20 μg ml⁻¹, respectively; (c) comparison of the relative catalytic activities of different reaction systems; (d) relative catalytic activities of the Au@NH₂-MIL-125(Ti) nanohybrid catalyst versus various pH values.

To gain a better understanding of the catalytic mechanism of the peroxidase-like nanozyme, Au@NH₂-MIL-125(Ti) was further characterized as a biomimetic enzyme by an apparent steady state kinetic analysis with H₂O₂ and TMB as substrates, respectively. In order to understand the catalytic mechanism and acquire kinetic parameters, kinetic experiments were performed by varying the concentration of one substrate while keeping the concentration of another constant. The concentrations of the blue product were calculated from the obtained absorbances at 652 nm using a molar attenuation coefficient value of 39 000 M⁻¹ cm⁻¹. It could be seen in Table 1 that the average K_m value for H₂O₂ of the prepared Au@NH₂-MIL-125(Ti) nanohybrid is 1.75 mM which is lower than that of horse radish peroxidase (HRP) (3.7 mM). On the other hand, the average K_m value for TMB was calculated to be 0.20 mM, which is also lower than the value for HRP (0.43 mM). The calculated average K_m values suggest that this nanostructure has higher substrate affinities than the HRP enzyme.¹² It is worth noting that, as is observed for natural HRP, the proximate parallel double reciprocal lines shown in Fig. 5b and d verify its ping-pong mechanism.^6,36

Table 1 Kinetic parameters of Au@NH₂-MIL-125 and horseradish peroxidase (HRP) with TMB and H₂O₂ as substrates, respectively

Substrate	Catalysts	K m/mM	Substrate	K m/mM
TMB	HRP	0.434^6	H2O2	3.7^12
	Au@NH2-MIL-125(Ti)	0.20 ^this work		1.75 ^this work
	Fe3O4	0.098^6		154^12
	MoS2-PPy-Pd	0.93^22		6.4^22
	Co3O4@CeO2	0.046^20		0.35^20

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Fig. 5 Steady-state kinetic assays for Au@NH₂-MIL-125(Ti). Experimental conditions: catalyst concentration 20 μg ml⁻¹, acetate buffer solution 5 ml and pH = 4. (a) TMB concentration was fixed at 0.1, 0.2, and 0.3 mM, respectively, while the concentrations of H₂O₂ varied from 1 to 10 mM; (c) H₂O₂ concentration was fixed at 1, 5, and 10 mM, respectively, while the concentrations of TMB varied from 0.1 to 0.5 mM; (b and d) the corresponding double reciprocal plots of H₂O₂ and TMB.

It is commonly known that the mechanism in peroxidase-like nanozymes stems from their ability to catalyze the decomposition of H₂O₂ to generate ˙OH and ˙O₂⁻.^36–39 Therefore, herein, radical trapping agents were used to elucidate the catalytic mechanism of Au@NH₂-MIL-125(Ti). As shown in Fig. 6, typical scavengers, terephthalic acid (TA) and isopropanol (TPA) as ˙OH scavengers, and benzoquinone (BQ) as a ˙O₂⁻ scavenger, were added into the H₂O₂  +  TMB + Au@NH₂-MIL-125(Ti) system while keeping other conditions fixed. As expected, the absorbance at 652 nm decreased sharply with the addition of BQ as a radical trapping agent, while only a slight change in adsorption at 652 nm can be observed in the system by adding TA or IPA. Thus, it can be deduced that the strong oxidizing ˙O₂⁻ radicals generated during the reaction between Au@NH₂-MIL-125(Ti) and H₂O₂ played major roles in the catalytic reaction. TMB can be oxidized to be the corresponding blue oxidation product by the generated ˙O₂⁻ radicals.

Fig. 6 Relative activity (%) of the H₂O₂  +  TMB + system in the absence (blank) or presence of radical scavengers (TA, IPA and BQ).

As we know, H₂O₂ plays an important role in biological systems, clinical diagnosis, environment, and food manufacturing.^1,4,5,40 Therefore, the sensitive detection of H₂O₂ is very important and has attracted much attention. It has been well established that the color and absorbance change of TMB oxidation catalyzed by peroxidase-like nanomaterials such as Au@NH₂-MIL-125(Ti), which has been proved to be a superior peroxidase-like nanozyme, are reported to be H₂O₂ concentration-dependent. Therefore, Au@NH₂-MIL-125(Ti) was first used for the sensitive detection of H₂O₂ by a colorimetric strategy. As clearly shown in Fig. 7a, the absorbance values at 652 nm of the reaction system increase with the increase of the H₂O₂ concentration. Typically, the absorbance values are in a fixed proportion to the H₂O₂ concentrations ranging from 2 to 10 μM. Accordingly, the detection limit is calculated to be as low as 0.24 μM (S/N = 3), which is lower than those by other methods (shown in Table S1, ESI†). All the above results indicate that the developed nanozyme-based sensor has great potential for quantitative detection of H₂O₂ with ultrahigh sensitivity.

Fig. 7 (a) The dose–response curve with regard to the H₂O₂ detection; (b) the corresponding calibration curve.

Bio-thiols, such as cysteine, play key roles in the process of reversible redox reactions in physiological systems and the presence of abnormal contents will lead to various diseases.^41–43 Therefore, it is vital important to develop sensitive sensing methods for cysteine. As is known to all, TMB can be oxidized into a blue product by H₂O₂ in the presence of a catalyst. However, the oxidation can be totally or completely reduced by adding cysteine due to its antioxidant properties. Furthermore, the enzymatic activity can be inhibited by cysteine due to its strong bonding capacity onto the Au surface to mask the active sites. Both effects lead to the reduction of TMB oxidation and lighten the blue color of the reaction system. Based on the above discussions, a colorimetric strategy for cysteine detection has been developed. Accordingly, the ΔA values at 652 nm of the reaction solution, catalyzed by the Au@NH₂-MIL-125, were determined in the presence of cysteine with varying concentrations ranging from 1 μm to 1000 mM. As displayed in Fig. 8a, it is obvious that the ΔA values increase along with the increasing cysteine concentration. Moreover, a good linear trend is achieved in the range of 1–10 μM. Accordingly, the detection limit (LOD) for cysteine sensing is calculated to be 0.14 μM (S/N = 3), which is lower than or at least comparable to previously reported colorimetric methods for cysteine detection (shown in Table S2, ESI†). This linear range and detection limit for cysteine detection are reasonable. Therefore, Au@NH₂-MIL-125 could be used as an effective peroxidase-like nanozyme and has potential for application in colorimetric detection of cysteine.

Fig. 8 (a) Curve graph of absorbance versus concentrations of cysteine and (b) the corresponding linear calibration plot for cysteine. Experimental conditions: TMB (0.1 mM), H₂O₂ (5 mM), catalyst suspension (20 μg ml⁻¹), and reaction time (10 min).

Specificity is another crucial index in cysteine detection. Therefore, we further studied the selectivity of detection towards other biomolecules. Fig. 9 shows the inhibition effects of some typical common amino acids and glucose on the TMB oxidation. Obviously, in the presence of cysteine, the ΔA values are much higher than those of other used interferences except for glutathione, indicating that cysteine shows an efficient inhibition effect on the reaction system. On this basis, we constructed a rapid and sensitive colorimetric method for cysteine detection.

Fig. 9 Inhibition effects of various amino acids and glucose on TMB oxidation. Experimental conditions: TMB (0.1 mM), H₂O₂ (5 mM), catalyst dispersion (20 μg ml⁻¹), amino acid and glucose concentration (100 μM), and reaction time (10 min).

Considering that a variety of diseases are associated with cysteine, it is of importance to explore the reliability of the developed assay under physiological conditions.^44–48 It is well known that human blood plasma contains high concentrations of cysteine (165–335 μm),⁴⁴ homocysteine (9–13 μM)^45,46 and glutathione (14–20 μm).⁴⁷ Herein, the practicality of the provided assay for cysteine detection was evaluated with fetal bovine serum as the sample by applying a standard addition method.⁴⁸ The standard curve (Fig. S1, ESI†) is obtained by conducting cysteine detection in the as-prepared serum sample according to the developed assay, where cysteine with different concentrations was spiked into the diluted fetal bovine serum and human serum and then quantified according to the procedure discussed above. The recoveries of the cysteine are found to distribute within the range from 93.8% to 103.5% with RSD values ranging from 3.2% to 6.1% as shown in Table 2. The desirable results validate the reliability and practicality of the provided colorimetric assay for cysteine analysis in real samples. Taken together, we established a reliable colorimetric assay that can be readily applied in biological applicability by measuring cysteine in fetal bovine serum and human serum.

Table 2 Determination of cysteine in fetal bovine serum

Samples	Added/μM	Found^a/μM	Recovery%	RSD/%
a Mean of five determinations ± standard deviation.
Fetal bovine serum	4.0	3.754	93.8	3.23
	7.0	7.022	100.3	5.62
	9.0	9.320	103.5	6.13

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Mercury ions (Hg²⁺) can cause a series of serious health and environmental problems due to their high biological accumulation and toxic properties^49–54 Therefore, it is urgent to develop sensitive and selective detection strategies for Hg²⁺ in water and blood. Colorimetric assays have been proved to be a rapid, visual read-out and high throughput analysis method for Hg²⁺ detection. As discussed above, the catalytic activity of Au@NH₂-MIL-125(Ti) was greatly inhibited in the presence of cysteine at certain concentrations. As described in previous reports, Hg²⁺ has higher affinity towards –SH than that of Au, thereby the inhibited activity can be completely or partially recovered by the existence of various concentration of Hg²⁺.⁵⁴ Thus, herein, a colorimetric method for Hg²⁺ detection was developed using cysteine as an inhibitor. Although the catalytic activity of Au@NH₂-MIL-125(Ti) toward TMB oxidation can be fully inhibited by a higher concentration of cysteine, the recovery of catalytic activity was not easy in the presence of excess cysteine. Therefore, the detection of Hg²⁺ was carried out with 60 μM cysteine. As expected, along with the increasing concentration of Hg²⁺, the absorption values increased gradually and present a linear relationship ranging from 1 to 5 μM (Fig. 10a). The detection limit of Hg²⁺ calculated from the linear equation presented as y = 0.03023x + 0.21155 in Fig. 10b was determined to be as low as 100 nM (S/N = 3), which is much lower than the established maximum tolerant level of Hg²⁺ in blood (0.01 mg L⁻¹ and 500 nM).⁵⁴ As shown in Table S3 (ESI†), our results were compared with other previously reported Hg²⁺ detection methods. The linear trend and LOD value of this assay are comparable to some of previously reported fluorometric and colorimetric based assays.^50–58 However, a lower LOD value and improved sensitivity are desirable to meet the permissible value (0.006 mg L⁻¹ and 30 nM) for drinking water.⁵¹

Fig. 10 (a) Dose–response curve for absorbance at 652 nm vs. Hg²⁺ concentration and (b) the linear calibration plot for detection of Hg²⁺. Experimental details: TMB (0.1 mM), H₂O₂ (5 mM), catalyst dispersion (20 μg ml⁻¹), cysteine (60 μM), and reaction time (5 min).

As shown in Fig. 11, the selectivity for Hg²⁺ was determined against various other metal ions in the presence of cysteine. As we know, Hg²⁺ has a larger stability constant with thiol ligands than other metallic ions, including Ni²⁺, Co²⁺, Zn²⁺, Pb²⁺, and Zr⁴⁺, which is the prerequisite for the selective detection of Hg²⁺. However, obvious interference of some metal ions like Ag⁺, Fe³⁺, Cu²⁺ and Bi³⁺ was observed. Compared to the control test, the existence of such metal ions also enhances the absorbance value. This phenomenon can be ascribed to two possible reasons. Some ions like Cu²⁺ and Bi³⁺ also have high binding capacities compared to thiol ligands. Meanwhile, some ions like Ag⁺ and Fe³⁺ have intrinsic catalytic activity toward TMB oxidation. Therefore, to eliminate the potential interference from these ions, EDTA and chloride (molar ratio = 2 : 1) were used as masking agents. Notably, the interferences of these ions were almost eliminated completely in the presence of these two masking agents. Concurrently, the presence of Hg²⁺ can be detected sensitively without interference, even with the naked eye by the change of the color of the solution from colorless to blue.

Fig. 11 Absorbance of the TMB solutions before and after masking (0.5 mM EDTA, 1 mM NaCl) in the presence of 100 μM different metal ions.

The practical applicability of this colorimetric assay for the detection of Hg²⁺ in environmental water samples was further investigated. The standard addition method was used to detect the concentrations of Hg²⁺ in tap water from Dalian Minzu University. Hg²⁺ at different concentrations was spiked into the water samples and then quantified according to the procedure discussed above. The results are listed in Table 3. It can be observed that the recovery values of Hg²⁺ in the water samples are between 91.56 and 106.9%. The relative standard deviations (RSD, n = 5) at each concentration level are all less than 3.03%. The results indicate that this assay can be used for the qualitative analysis of Hg²⁺ in environmental water samples with good recovery and precision.

Table 3 Determination of Hg²⁺ in the environmental water sample

Samples	Added/μM	Found^a/μM	Recovery%	RSD/%
a Mean of five determinations ± standard deviation.
Tap water	3	3.123	104.1	3.03
	6	5.493	91.56	2.03
	9	9.629	106.9	2.53

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4. Conclusion

In summary, we have fabricated an Au@NH₂-MIL-125(Ti) composite by an impregnation method followed by a borohydride reduction method, in which small and uniform Au metallic nanoparticles were successfully deposited on the surface and entrapped into the nanocages of NH₂-MIL-125(Ti). The resulting nanohybrid nanozyme demonstrated an obviously better peroxidase-like catalytic activity than single Au NPs and NH₂-MIL-125(Ti). Its superior peroxidase-like activity was attributed to the synergistic effect of the small size of Au NPs and NH₂-MIL-125(Ti). Furthermore, the kinetic experiments revealed that the nanozyme has lower K_m values for both TMB and H₂O₂ compared to natural HRP, indicating its higher affinity to both substrates. Motivated by these results, a multifunctional platform for the facile colorimetric sensing of hydrogen peroxide, cysteine and Hg²⁺ was developed. The detection limits for H₂O₂ and cysteine were as low as 0.24 μM and 0.14 μM, respectively. By introducing EDTA and chloride as masking agents, Hg²⁺ can be selectively detected with a very low detection limit down to 100 nM without interference of many other metal ions. Furthermore, the practical applicability for detection of both cysteine in fetal bovine serum and Hg²⁺ in tap water was confirmed. In summary, the proposed multifunctional platform is sensitive, fast and selective, and shows great promise for practical applications in biosensing and biotechnology.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21473190, 21203017), the Liaoning Natural Science Foundation of China (20180551022) and the Fundamental Research Funds for the Central Universities.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb02183c

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