VOx Quantum Dots with Multienzyme-Mimic Activities and the

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VOx quantum dots with multienzyme-mimic activities and the application in constructing a three-dimensional (3D) coordinate system for accurate discrimination of the hydrogen peroxide over a broad concentration range Lei Huang, Yusheng Niu, Ronggui Li, Haozhong Liu, Yao Wang, Gengfang Xu, Yang Li, and Yuanhong Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05923 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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Analytical Chemistry

VOx quantum dots with multienzyme-mimic activities and the application in constructing a three-dimensional (3D) coordinate system for accurate discrimination of the hydrogen peroxide over a broad concentration range Lei Huang‡,a, Yusheng Niu‡,a, Ronggui Lia, Haozhong Liub, Yao Wanga, Gengfang Xua, Yang Lic, Yuanhong Xu*,a College of Life Science, Qingdao University, Qingdao 266071, China. of Urology, Key Laboratory of Urinary System Diseases, the Affiliated Hospital of Qingdao University, Qingdao 266003, China c College of Materials Science and Engineering, Qingdao University, Qingdao 266071, China a

b Department

ABSTRACT: The construction of efficient nanozyme with multienzyme activities in a simple way is vital for the wide biological and chemical applications. Generally, the mimic enzyme activities depend on their sizes, surface states and materials types. Quantum dots (QDs), one type of zero-dimensional nanomaterials, are much appealing due to their abundant catalytically active surface deficiency. The vanadium oxide (VOx) is one special transition metal oxides possessing different valence states. Inspired by these views, we synthesized VOxQDs herein via a one-pot top-down ethanol-thermal method using bulk VO2 as the precursor. The VOxQDs showed not only oxidase- and peroxidase-like activities in ethanol as the main background solution (ethanol-BGS), but also exhibited additional superoxide dismutase mimetic activity in phosphate buffer solution. Furthermore, the TMB-VOxQDs system in the ethanol-BGS produced three distinct colors in the presence of hydrogen peroxide (H2O2) at three different concentration gradients (10-90 μM, 0.1-10 mM and 20-100 mM). Accordingly, we constructed a three-dimensional (3D) coordinate system (3D-CS) by using the three variables: the initial velocities, the maximum absorption values and the visual colors of the enzymatic reaction system. As a result, the rapid detection of H2O2 can be achieved while effectively avoiding the faked appearance due to the inhibition effects to the enzymatic system at too high H2O2 concentration. The applicability of the VOxQDs based 3D-CS was further proved via the facile and accurate H2O2 assays in three different practical samples.

Nanomaterials with intrinsic enzyme-mimic activity (nanozymes) have been attracting great interest due to their remarkable superiorities including low cost and high stability in catalytic reactions. They are suggested to be promising substitutes for the natural enzymes in various bio-/chemapplications.1 Vanadium oxide nanomaterials such as V2O5 nanowires,1,2 VOx nanoflakes,3 V2O5 nanoparticles4 have also been reported to possess bionic enzyme properties. But most of the reports are limited to the study of their peroxidase (POD)like activities, the researches of other ones such as superoxide dismutase (SOD), oxidase (OXD) and catalase (CAT) of vanadium oxides are much fewer.5 Since multienzymes are important in many bio-/chem- processes such as antiinflammatory,6,7 antibacterials,8-10 and biosensing,11 many researchers have been involved in combining individual VOx nanozyme with other nanomaterials or matrix to form the nanocomposites with multienzyme activities. For example, a novel antioxidant-defense multienzyme, V2O5@polydopamine@MnO2, can be produced using polydopamine as the linker to combine the glutathione peroxidase (GPx)-like V2O5 nanowire and MnO2 nanoparticles with mimic SOD and CAT activities.12 However, their preparation was still relatively complex. Thus it is still urgent

to explore a VOx nanozyme possessing multienzyme-mimic activities via a relatively simple strategy. Quantum dots (QDs) are one distinct nanoparticle with sizes of smaller than 100 nm, which showed many superiorities including unique optical feature, better dispersibility and larger specific surface area compared with their bulk form, because of the surface functional groups, quantum confinement and edge effects.13 Generally, the surface charges, oxygen vacancies, size, shape, and crystal facets are important for the mimic enzyme activities of inorganic metallic oxides.14,15-17 VOxQDs were found with different valence states and mixed phases of vanadium oxide in our previous work.18 These findings inspired us to design a multienzyme based on the VOxQDs. In addition, the previous reported VOxQDs were prepared via a dimethyl sulfoxide (DMSO)-based agitation method. Since DMSO is relatively toxic at room temperature.19 It is still in great demand to develop a more simple and environmental-friendly way to prepare the VOxQDs. Hydrogen peroxide (H2O2) is existing in diverse important biological processes in vivo or in vitro.20 It also plays significant roles in the photochemical reaction and the redox

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reaction in the natural environment, affecting redox chemistry and thedistribution of the biologically active metals including manganese, copper, and iron in natural surface waters.21 Various strategies can be used for the H2O2 assays, including fluorometric,22 chemiluminescent,23 spectrophoto-metric,24 and electrochemiluminescent25 ones. Therein, the chromogenic reaction based on either the natural enzyme or nanozyme was considered to be a relatively visible method with simple operation, quick response and high accuracy.26-28 Also, both natural or biomimetic POD catalyzed reaction might be inhibited at the high concentration of H2O2.29 Accordingly, a high concentration of H2O2 and a low one might produce the same color reaction and initial reaction speed. However, to detect the H2O2 concentration of an unknown sample based on the TMB chromogenic reaction, it has to measure the initial reaction speed to bring into the Michaelis-Menten equation with known Km and Vmax, or directly assess the corresponding concentration according to the color intensity. Thus, it's difficult to accurately distinguish the H2O2 concentration if the inhibition reaction might occur, which is also an overlooked problem in previously reported enzyme-based H2O2 assays. To overcome the challenges mentioned above, the VOxQDs were synthesized via a one-pot ethanol-thermal method using vanadium dioxide (VO2) as the precursor. The multienzyme activities, including SOD, OXD, and POD of the as-prepared VOxQDs were investigated in different background solutions (BGS) of both ethanol and phosphate buffer solution (PBS), respectively. A variety of characterizations have been carried out to reveal the enzymatic reaction processes and mechanisms. In addition, different color reactions of VOxQDs suspension were produced upon the addition of different concentrations of H2O2. Accordingly, we constructed a threedimensional coordinate system (3D-CS) by using three factors: the initial velocities, the maximum absorption peak values and the colors of the reaction suspensions. Accordingly, the 3D-CS could be used in the assays of H2O2 at varied concentrations. It is not only feasible for the enzyme reaction in accordance with the Michaelis-Menten equation but also applicable for the inhibited one at too high H2O2 concentration. The practical applicability of the 3D-CS has also been verified by the H2O2 assays in Qingdao's spring rain, the surface water of Dagu River, Qingdao and aquariums water which contaminated with algae, respectively.

EXPERIMENTAL SECTION Materials. Vanadium oxide (VO2), europium chloride (EuCl3·6H2O) and Tetracycline hydrochloride were purchased from Shanghai Macklin Biochemical Co. Ltd., China. Ethanol, hydrogen peroxide (H2O2), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O, ≥ 99.0 %) and monobasic sodium phosphate dihydrate (NaH2PO4·2H2O, > 99 %) were purchased from Sinopharm Chemical Reagent Co. Ltd., China. 3,3’,5,5’-tetramethylbenzidine (TMB) was purchased from the Shanghai YuanYe Bio-Technology Co. Ltd. Horseradish peroxidase (HRP, >250 U/mg), terephthalic acid (TA), riboflavin, methionine and NBT were obtained from Shanghai Macklin Biochemical Co. Ltd.Ultrapure water (18.2 MΩ cm−1) was obtained from a Milli-Q ultrapure system (Qingdao, China). All reagents were of analytical grade and used without any further purification. Apparatus. The photoluminescence (PL) spectra were recorded on an FS5 Spectrofluorometer (Edinburgh, United

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Kingdom). The excitation/emission slits were set at 4.0 nm × 4.0 nm. The UV absorption spectra were gained from a Mapada UV-6300 spectrophotometer (Shanghai, China). Xray photoelectron spectroscopy (XPS) data were carried out on an ESCALab 220i-XL electron spectrometer (VG Scientific, West Sussex, the United Kingdom) using 300 W Al Kα radiation. The morphology of the VOxQDs was characterized by a JEM-2010 transmission electron microscope (JEOL Ltd., Japan). Synthesis of VOxQDs. The VOxQDs were synthesized via a one-pot ethanol-thermal method. 0.2 g VO2 samples were added into a reaction kettle containing 20 mL ethanol and then were solvothermally treated at 200 ℃ for 10 h. After cooling to the room temperature, the VOxQDs were obtained upon collecting the supernatant of the products centrifuged at 12000 rpm for 10 min. The OXD- and POD-like activities of VOxQDs in ethanol solution. The OXD-mimic behavior of VOxQDs was investigated by oxidizing TMB without H2O2. In detail, different concentrations of TMB (50, 100, 200, 300, 400 and 500 μM) were added into a 3 mL reaction system which contained 1 mL VOxQDs suspension. The mixed suspensions were incubated at 25 °C for 5 min. Then we measured the UVVis absorbance at 600 nm of the corresponding mixtures. The POD-mimic activity of VOxQDs was measured by oxidizing TMB within H2O2. For the chromogenic reaction of TMB at different concentrations of H2O2, the samples were prepared by mixing 1 mL VOxQDs (10 μg/mL, dissolved in ethanol), 1 mL TMB (300 μg/mL, dissolved in ethanol) and 1 mL H2O2 (diluted by deionized water into different concentration gradients), thus the ethanol/water volume ratio was kept at 2:1. The kinetic analysis was carried out immediately at 900 nm or 600 nm. The FL measurement of •OH generation in different reaction systems was performed as follows: 0.5 mM TA was added to the reaction solution containing 10 mM H2O2 and 5 μg/mL or 10 μg/mL VOxQDs, and be followed with incubation for 5 min. In order to investigate the combined actions of the OXD- and POD-like activities, different concentrations of TMB (50, 100, 200, 300, 400 and 500 μM) were added into 3 mL reaction system which contains 1 mL VOxQDs and 100 μM H2O2. The measurement of the SOD-like activity of VOxQDs in PBS. The SOD mimics activity of VOxQDs was proved by the consumption of superoxide anion radicals (O2-) and the generation of H2O2 and O2. The consumption of O2-was measured by the inhibition towards the photoreduction of nitro blue tetrazolium (NBT).12 The mixture containing NBT (75 μM), methionine (0.013 M), riboflavin (20 μM) and 0, 1.25, 2.5, 5, 7.5, 10, 15, 20 μg/mL VOxQDs were prepared with 50 mM PBS (pH 7.4). The mixture solutions were exposed to ultraviolet illumination for 10 min at 25 °C. After irradiation, the absorbance was studied at 560 nm immediately. A blank control without UV radiation and a group without VOxQDs upon UV radiation were also measured. The formation of H2O2 was studied using the europium tetracycline (EuTc) based FL assay. MOPS buffer was used to avoid the interference of the citrate and phosphate to the EuTc FL intensity.30 Preparation of EuTc solution was conducted as follows: 115.3 mg of EuCl3·6H2O and 50.5 mg of tetracycline hydrochloride were dissolved in 50 mL of MOPS (10 mM, pH=7.4, 25 °C), respectively. Then the two solutions both in 5


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mL were mixed and then diluted by MOPS buffer to 50 mL. Different concentrations (0-20 μg/mL) of VOxQDs were added to 3 mL EuTc solution to measure the FL intensity, respectively. The formation of elemental oxygen with different VOxQDs concentrations (0, 0.05, 0.09, 0.13, 0.17, 0.2 mg/mL) was monitored using an oxygen sensitive electrode. The oxidation of TMB with the involvement of multienzyme-like catalyst and the pH and resistance properties of VOxQDs. The oxidation of TMB with the involvement of multienzyme-like catalyst was detected with the concentration of H2O2 fixed (100 μM) and TMB varied (0300 μM) in 50 mM PBS at pH=7 and pH=4, respectively. The pH-depended enzyme-like activity of VOxQDs was measured in 50 mM PBS with different pH values (pH1-pH12). In detail, different concentrations of TMB (from 100 μM to 500 μM) were added to 3 mL 50 mM PBS at different pH containing 100 μM H2O2 and 500 μL 10 μg/mLVOxQDs at 25 ℃. The temperature depended enzyme-like activity of VOxQDs was measured with the same sample system as pH depended study while the temperature varied from 5 to 85 ℃ in 50 mM PBS at pH=4. Detection of H2O2 in natural waters by the 3D-CS method. The natural waters were collected in glass bottles with Teflon-lined caps and stored at 4 ℃ in the refrigerator immediately upon receipt. Before testing, the samples were filtered through a 0.45-pm filter. Then 1 mL VOxQDs (10 μg/mL, dissolved in ethanol), 1 mL TMB (300 μg/mL, dissolved in ethanol) and 1 mL sample solutions were mixed and incubated at 25 °C for 5 min for UV-Vis detection and kinetic analysis.

RESULTS AND DISCUSSION Synthesis and Characterization of the as-prepared VOxQDs. As can be seen in Figure 1A, bulk VO2 powder was firstly ethanol-thermally treated at 200 °C for 10 h. The asobtained products were centrifuged at 12000 rpm for 10 min. The supernatant of the products was considered to be the VOxQDs suspension, which was characterized by the transmission electron microscopy (TEM) (Figure 1B) for morphology study. As shown, it exhibited well-dispersed nanodots with lateral diameter distribution ranging between 0.86 and 6.13 nm (Figure 1C). The mean size was 3.15±0.3 nm as calculated from 100 particles in the TEM image. Meanwhile, highly paralleled fringe of the nanodots were observed in the high-resolution TEM (HRTEM). Lattice interspace of about 0.202 nm (left inset in the Figure 1B), 0.27 nm (Figure S1A) and 0.39 nm (Figure S1B) were observed in the crystal structures of the VOxQDs, which was in accordance with the previously reported diffraction planes of VO2 (JCPDS 81-2393), V2O3 (JCPDS 76-1043) and V2O5 (JCPDS 85-2422), respectively.18,31,32 All these results confirmed the successful generation of the VOxQDs based on the simple ethanolthermal treatment. The optical properties of the as-obtained VOxQDs were studied by the UV-Vis absorption and FL emission spectra. As shown, it was colorless under daylight (left inset in Figure 2A) and bright blue under 365 nm UV irradiation (right inset in Figure 2A). As shown, the VOxQDs showed typical UV-Vis absorbance at 320 nm. Meanwhile, the optimum excitation and emission located at 360 and 450 nm, respectively.33

Figure 1. (A) Scheme of theVOxQDs generation through the ethanol-thermal method. (B) TEM and HRTEM (inset) images and (C) the corresponding lateral size distributionsof the asprepared VOxQDs.

We further collected the 3D FL spectra of the VOxQDs under a variety of excitation wavelengths (Figure 2B). The emission peaks shifted from 430 to 460 nm with the gradually increasing excitation wavelengths from 330 to 400 nm, confirming an excitation-dependent behavior of the VOxQDs FL emission.34 The location in (0.21, 0.22) upon the excitation wavelengths at 360 nm in the 1931 CIE chromaticity diagramagain confirmed the blue emission of the as-prepared VOxQDs under UV irradiation (Figure 2C). All the results mentioned above indicated the successful preparation of VOxQDs with excellent FL, which should be due to the quantum confinement and edge effects generated in the ethanol-thermal process.13

Figure 2. (A) The UV-Vis absorbance as well as the maximum excitation (EX) and emission (EM) wavelength in the FL spectra. The insets in A) Photographs of the VOxQDs suspension in daylight (left) and under exposure of 365 nm UV light (right).



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(B)The 3D FL spectra at gradually mounting excitation wavelengths. (C) The CIE 1931 chromaticity chart at excitation of 360 nm. The characterizations of the as-obtained VOxQDs: (D) The XRD pattern; (E) The XPS spectra and (F) the corresponding narrow scan spectra of V-2p.

Figure 2D shows the crystalline structure of (a) the precipitation after ethanol-thermal treatment and (b) the asprepared VOxQDs via XRD characterization. For the precipitation, the main peak at 2θ = 27.7° is attributed to V2O5 (JCPDS 85-2422), the peaks at 25° and 32.8° are assigned to V2O3 (JCPDS 76-1043) and the ones at 40°, 45.3° are ascribed to VO2 phase. Compared with the precipitation, VOxQDs showed the main peak broadens and crosses between 20° to 50°, the mixed valence state can be deduced by combining with the HRTEM images. The weakening and broadening of the peaks after solvothermal action indicated that there were many defects on the surface of VOxQDs, which played an important role in the exhibition of enzyme activity.3 X-ray photoelectron spectroscopy (XPS) was further performed to study the composition and functional groups on the surface of VOxQDs. In the survey XPS spectra, the four obvious peaks at 532, 517, 400, 285 eV resulted from the elements of O, V, N and C, respectively (Figure 2E).35 While the fitted XPS spectrum of V-2p showed a tiny peak of V3+ (V2p1/2at 524.2 eV) and big peaks of V4+ and V5+, being ascribed to the V2p3/2 at 516.5 and 517.7 eV, respectively (Figure 2F).36 As a result, the coexistence of the multiple phases in the as-obtained VOxQDs can be further confirmed by the XPS analysis. The OXD- and POD-like activities of VOxQDs in ethanol background solution. The OXD- or POD- like activities of VOxQDs in ethanol background solution (ethanol-BGS) were firstly investigated using H2O2 and/or TMB as substrates (Figure 3A). The ethanol-BGS containing an equal volume of three reaction solutions (VOxQDs suspension, TMB dissolved in ethanol and H2O2 aqueous solution) to ensure the convenience of operation, thus ethanol/water volume ratio of 2:1 was used throughout the test. Firstly, H2O2 with TMB or VOxQDs alone did not produce obvious color absorbance (curves and insets a, b in Figure 3A). While dark blue color was observed when 100 μM H2O2 (added concentration, similarly hereinafter in the ethanol-BGS system) was mixed with both TMB and VOxQDs (curve and inset d in Figure 3A). The color change was ascribed to the TMB oxidation in the presence of H2O2 catalyzed by the VOxQDs.37 These results indicated that the intrinsic POD-like catalytic activity of VOxQDs toward TMB. Generally, the POD-like activity of VOxQDs can catalytically decompose H2O2 to hydroxyl radicals (•OH).38 Accordingly, TA was applied as a fluorescent probe to confirm the •OH generation during the interaction between the VOxQDs and H2O2,39,40 since TA can specially react with •OH to form 2-hydroxy terephthalic acid (TAOH), which showed a distinct FL signal at 435 nm. As shown in Figure S2, the FL of TA was significantly enhanced in the presence of both the VOxQDs and H2O2, confirming that the conversion H2O2 into •OH due to the POD-like activity of the VOxQDs.

Figure 3. (A) Time-dependent absorbance changes at 900 nm in different reaction systems: VOxQDs + H2O2, TMB + H2O2, VOxQDs + TMB and TMB + VOxQDs + H2O2 in neutral ethanolBGS. (B) and (C) are catalytic oxidations of various TMB concentrations in the presence and absence of 100 μM H2O2, respectively. (D) Comparison of OXD-like catalytic activity and the collaboration of OXD- and POD-like activities of VOxQDs in TMB oxidation.

Moreover, the VOxQDs could also render the TMB solution a light blue color with the main absorbance at 600 nm (curve and inset c in Figure 3A) in the absence of H2O2. This result indicated the VOxQDs possessed intrinsic OXD-mimic activity, which should be attributed to the production of reactive oxygen species (ROS) from O2 upon the catalytic oxidation effects of the VOxQDs. Thus, it can be proposed that the VOxQDs showed bienzyme activities of OXD and POD simultaneously in the presence of H2O2. To confirm this proposal, we collected the UV-Vis absorption and the visual color of the VOxQDs upon the addition of TMB at different concentrations in the presence (Figure 3B and Figure S3A, optical image a in Figure 4) or absence (Figure 3C and Figure S3B, optical picture b in Figure 4) of 100 μM H2O2. The absorbance of the two samples was both enhanced with the incremental concentrations of the TMB (Figure 3B and 3C). The colors were also deepened correspondingly. In addition, the absorbance and color of the VOxQDs with H2O2 was higher and deeper than those without H2O2 at each given TMB concentration, respectively (Figure 3D). This phenomenon showed that the catalytic ability of VOxQDs to oxidize TMB was much stronger after adding H2O2. This is because in the absence of H2O2, the VOxQDs showed only OXD-like catalytic ability to TMB, while the catalytic oxidation was significantly enhanced due to the co-participation of POD-like catalysis in the presence of H2O2. As a result, the VOxQDs were confirmed to be with bienzyme activity in the ethanolBGS. Interestingly, it was observed that the color of the VOxQDsTMB system changed upon the variation of the H2O2 concentration, especially showed different series in the different concentration gradients. It was mainly dark blue in the H2O2 concentration of 10-90 μM (optical images c in Figure 4), while became gray-green at 100 μM and then deepened gradually to brown till to 10 mM (optical images d in Figure 4).


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Analytical Chemistry Table 1. Comparison of the kinetic parameters of VOxQDs while the reaction solutions were generated different colors.

Substrate

Substrate fixed

Km(mM)

Vmax (10-8M*s-1)

Brown

H2O2

TMB (400 μM)

0.274 (± 0.016)

6.39 (± 0.09)

Blue

H2O2

TMB (400 μM)

0.008 (± 0.001)

2.17 (± 0.03)

Colors

The values are shown as the mean ± SD (n = 3).

Figure 4. Schematic illustrations for the reaction process of VOxQDs in ethanol and PBS as the BGS, respectively.

Furthermore, the solution changed to yellow with the ongoing increase of the H2O2 concentration from 20 to 100 mM (optical images e in Figure 4). The different color of the VOxQDs-TMB system should result from the various oxidized state of TMB with different concentrations of H2O2.37,41

The UV-Vis spectra were measured in the presence of three different concentration gradients of H2O2. The UV-Vis absorbance increased with the incremental H2O2 in micro level concentration gradients then reached to the threshold and decreased in millimol level. The maximum absorption peaks of the VOxQDs-TMB system were 600 nm (Figure 5A) and 900 nm (Figure 5B) in the presence of H2O2 concentration of 10-80 μM and 20-100 mM, respectively. In addition, the absorbance showed a trend of first sharp increase and then weak change with the increasing reaction times (Figure 5C). The corresponding time-dependent UV-Vis absorbance changes (Figure 5D-5F) showed the same trend as the UV-Vis spectra. The different absorption behaviors were consistent with the three different colors in the different H2O2 concentration spans (Figure 6). As can be seen in Figure 6A and 6B, the reaction rates and the absorbance of the system showed an upward trend with the increasing H2O2 concentrations ranging from 5 μM to 500 μM, while decreased sharply after H2O2 concentration at 10 mM. The decreased reaction rates and absorbance should be due to the inhibition effects of H2O2 at high concentration to the enzyme-like activity of the VOxQDs.29 By the linear fitting of Lineweaver-Burk double reciprocal plots of the Michaelis-Menten equation (Figure 6C), the Vmax and Km were measured based on the MichaelisMenten equation with the fixedTMBconcentrationand H2O2 changed from 10 μM to 900 μM (Table 1):42 V=(Vmax



[S])/(Km + [S])

V: Initial reaction rate Vmax: Maximum reaction rate [S]: Substrate concentration Km: Michaelis constant

Figure 5. (A) and (B) are UV-Vis spectra of VOxQDs-TMB system with different concentrations of H2O2. (C) The UV-Vis absorbance of the VOxQDs-TMB system contained 50 mM H2O2 with the increasing reaction times. (D) (E) and (F) are timedependent absorbance changes of the VOxQDs-TMB system with varying concentration gradients of H2O2. The TMB concentration was 400 μM.

However, the Michaelis-Menten equation was not applicable to the UV-Vis absorbance of the VOxQDs-TMB system at too high H2O2 concentration above 1 mM. In addition, two linear relationships can be obtained for the UVVis absorbance of the VOxQDs-TMB system against the H2O2 concentration ranging from 10 μM to 70 μM and 20 mM to 100 mM, respectively (Figure S4). Since no typical maximum absorption peak was presented in the H2O2 concentration of 0.1-10 mM, the corresponding linear relationship was difficult to be obtained. As can be seen, neither the Michaelis-Menten equation nor the fitted linear relationship between absorbance and concentration of H2O2 was able to cover the full H2O2 concentration for the further assays of unknown H2O2. As shown, besides the maximum absorbance values and the initial



reaction velocities, the as-prepared VOxQDs herein as nanozyme can additionally lead to three different series corresponding to three different concentration ranges, which can be used to recognize the inhibition effect of H2O2 at too high concentration. Thus, it can be used to supplement the unmeasured maximum absorbance H2O2 at the concentration of 0.1-10 mM. Accordingly, we defined the maximum absorbance values of the VOxQDs-TMB system in different H2O2 concentrations as X-axis, their initial reaction velocities as Y-axis, and the three color series in the three different concentration gradients as Z axis to build 3D-CS (Figure 6D). One H2O2 concentration can be recognized via the three variable parameters. Thus, the three variable parameters were collected for H2O2 at a wide variety of concentration as listed in Table S1. As shown in Figure 6D, different coordinate points were corresponding to the different concentrations of H2O2. For example, (1.15, 1.89, 2) represented 50 μM H2O2, (0.91, 0.19, 1) corresponded to 50 mM H2O2, which can be used for assays of samples with an unknown concentration of H2O2.

Figure 6. (A) Absorbance values of maximum absorption peaks and (B) Initial reaction velocities in the presence of varying concentration gradients of H2O2. (C) The linear fitting of Lineweaver-Burk double reciprocal plots obtained by MichaelisMenten equation with fixed TMB concentration and varied H2O2 concentration. (D) Three-dimensional simulation of H2O2 concentration detection by the coordinate method, different points on the surface represent different concentrations of H2O2.The values are presented as the mean ± SD (n = 3).

The multienzyme-mimic activities of the VOxQDs in PBS. The enzyme-like catalytic activities of the VOxQDs were also studied using PBS as BGS. The POD- or OXD-mimic activities of the VOxQDs in PBS can be confirmed using H2O2 and/or TMB as substrates (Figure S5A), and in hypoxia, their catalytic ability was reduced significantly, as shown in Figure S5B, further indicated that the VOxQDs exercised the function of OXD. In addition, it was found the as-prepared VOxQDs also showed the SOD mimetic activity, which can be verified by measuring the reaction products. At first, as a vital antioxidant enzyme, the SOD can catalyze the disproportionation reaction of transforming superoxide anion radicals (O2-) to O2 and H2O2.43 As shown in Figure 7A, within the methionine, riboflavin and nitrotetrazolium blue chloride, an obvious absorbance signal at about 600 nm was obtained after UV irradiation,44 due to the generation of the

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high-level O2-. When the VOxQDs were present, the signal decreased dramatically due to the consumption of O2-.12 As shown in Figure S5C, the scavenging ability enhanced with the increasing VOxQDs concentration. Subsequently, the formation of H2O2 was studied using the europium tetracycline (EuTc) based FL assay.45 The EuTc alone showed low FL at 620 nm (with λex=405 nm), while H2O2 binding to its metal center could result in a significant enhancement of the FL intensity. Thus it can be used to directly measure the increase of the H2O2 concentration through the FL change of EuTc.46 The probe EuTc shows high specificity towards H2O2. Its FL could be affected by the ammonium and alkali ions, nor by the anions of nitrate, chloride, or sulfate at concentrations of up to 10 mM. However, the citrate and phosphate might result in an enhancement of the EuTc FL intensity.30 To avoid this interference, the 3-(N-morpholino) propane sulfonic acid (MOPS) buffers (pH=7.4) rather than PBS were used for H2O2 detection. With the increasing addition of VOxQDs into the EuTc solution in MOPS, the FL intensity of EuTc increased gradually (Figure 7B and Figure S5D).

Figure 7. (A) The absorbance and optical images (insets) of the superoxide radicals in diverse systems: (a) the blank control without UV radiation, (b) without 10 μg/mL VOxQDs upon UV radiation, (c) within 10 μg/mL VOxQDs upon UV radiation. (B) The FL spectra of different concentrations of VOxQDs in MOPS buffers that treated with EuTc solutions. (C) The FL intensity change-reaction time curves in the presence of VOxQDs at different concentrations. (D) UV-Vis absorption spectra with diverse concentrations of TMB (10, 20, 30, 50, 75, 100 and 150 μM) of the VOxQDs-TMB system.

In addition, we measured the dependence of the EuTc FL intensity on the different concentrations of VOxQDs upon the increasing reaction times (Figure 7C). As shown, both the increasing amount of VOxQDs and reaction time could lead to increasing formation of H2O2. Furthermore, the formation of oxygen, another vital reaction product in a SOD-mimic reaction, was measured via an oxygen-sensitive electrode. We can obtain that the higher the amount of the VOxQDs was added, the higher rates of the oxygen production was (Figure S6). Overall, the SOD mimetic activity of the VOxQDs was confirmed by the consumption of O2- and the generation of H2O2 and O2, respectively. Accordingly, it can be concluded that the VOxQDs were exhibiting multi-enzyme-like catalytic activities including OXD-, POD- and SOD-like ones. Meanwhile, the synergistic actions of the multienzymes of the VOxQDs could promote the oxidation of TMB in PBS, which can result in the oxidized


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TMB with absorbance at 652 nm (Figure 7D, Figure S3C and Figure S5E). The reaction processes should be as follows:47

The electron paramagnetic resonance (EPR) spectra also verified the formation of O2- and •OH during the reaction (Figure S7). The multifunctional enzyme-like activities of the VOxQDs should generate from the oxygen vacancies and the different valence states of vanadium. It was reported that the unevenly stripping from the bulks and interacted with solvent, there are some oxygen vacancies formation (Figure S8), which could result in the enzyme-like activities of the VOxQDs. In addition, the redox couples between different valence states (V3+, V4+ and V5+) would further contribute to the multienzyme activities of the VOxQDs.48-50 Then the dependences of the enzyme-like activities of VOxQDs and HRP on the temperature (Figure 8A) and the pH (Figure 8B) were tested. As can be seen, VOxQDs showed better temperature and pH tolerance than HRP. When the temperature exceeded 65 ℃, HRP was basically not active, but the VOxQDs showed high catalytic activity even at 80 ℃. These indicated that VOxQDs have better application prospects under high-temperature conditions compared with the natural HRP. The catalytic properties of VOxQDs firstly increased till pH 4.0 and dropped to nearly zero at pH above 8.0, which was very similar to that of HRP with the best pH value of 4.0, but VOxQDs showed better pH tolerance than HRP because when the pH reached 7.0, the catalytic activity of HRP decreased to zero, but VOxQDs still showed catalytic activity.

fixed concentration of H2O2 (100 μM) and varied concentration of TMB in PBS at pH=4.0 and pH=7.0, respectively. The values are shown as the mean ± SD (n = 3).

In addition, Figure 8C and Figure 8D showed the time dependences of the absorbance changes of TMB in different concentrations of TMB with fixed 100 μM H2O2 at pH=4.0 and pH=7.0, and the reaction rate at pH=4.0 was more higher than pH=7.0 when the same concentration of TMB was added (Figure S5F). Accordingly, the linear fitting of LineweaverBurk double reciprocal plots of the Michaelis-Menten equation can be obtained as shown in Figure 8E and Figure 8F, respectively. It can be obtained that the Vmax in the acid condition at pH=4.0 was about 5 times of that in the neutral PBS (pH 7.0) (Table 2). Above all, the POD-like functions of VOxQDs are very similar to but superior over those of natural HRP. Thus the VOxQDs are expected to play important roles in relatively more harsh environments. The multienzyme mimics of VOxQDs for the design of multienzyme system have potential application prospects in many aspects both in vitro and in vivo. For example, the OXD and POD mimic activities of VOxQDs can be used for biosensor,11,51 antibacterial treatment and promoting wound healing,8,52 oral diseases treatment,53,54 biofouling prevention,55,56 etc. The triple enzyme mimic of VOxQDs (OXD, POD and SOD) have the application prospects such as anti-inflammatory agents,57,58 anti-tumor59-61 and maintenance of the metabolic balance.62,63 Table 2. Comparison of the kinetic parameters of VOxQDs in PBS at pH=4.0 and pH=7.0.

VOxQDs

Substrate

Substrate added

Km(mM)

Vmax (10-9M*s-1)

pH=4.0

TMB

H2O2 (100 μM)

0.098 (± 0.012)

5.61 (± 0.08)

pH=7.0

TMB

H2O2 (100 μM)

0.217 (± 0.005)

1.2 (± 0.02)

The values are shown as the mean ± SD (n = 3).

Figure 8. (A) The temperature depended and (B) pH depended enzyme-like activity of VOxQDs and HRP with the TMB as substrate. Time-dependent absorbance changes in the presence of varying concentrations of TMB at (C) pH=4 and (D) pH=7 in PBS. (E) and (F) the linear fitting of Lineweaver-Burk double reciprocal plots obtained by Michaelis-Menten equation with

Detection of H2O2 in natural waters by three-dimensional coordinate method. In this study, the 3D-CS method which based on TMB chromogenic reaction was successfully used to detect the concentration regions of H2O2 in Qingdao's spring rain (Figure 9A), the surface water of Dagu river, Qingdao (Figure 9B) and aquariums water which contaminated with algae (Figure 9C), respectively. As shown in Figure 9D, the three reaction mixtures are blue, indicating that the concentrations of H2O2 in the solutions were less than 100 μM. Then the specific coordinates of the three solutions in the 3DCS were located in Figure 9E: A (0.72 ± 0.04, 1.03 ± 0.09, 2), B (1.02 ± 0.01, 1.32 ± 0.04, 2), C (1.11 ± 0.01, 1.80 ± 0.03, 2), respectively. Previously a set of coordinates of H2O2 concentrations have been obtained, as shown in Table S1. By comparing with known coordinates, it can observed that the H2O2 concentrations of A solution was from 5 μM to 10 μM, B was close to 10 μM, and C was at nearly 40 μM. Furthermore, the accuracy of this system was proved by the traditional Michaelis-Menten equation calculation. The linear fitting of



Lineweaver-Burk double reciprocal plots obtained by the Michaelis-Menten equation when the H2O2 concentration below 100 μM were gained as: Y=354.28X+46.11 (Y=1/V, X=1/[S]), the H2O2 concentrations in the three samples can be figured out: 6.66 μM, 11.54 μM and 37.83 μM for A, B and C, respectively. As can be seen, the results obtained by the 3DCS method without tedious calculation are very close to those calculated by Michaelis-Menten equation. Moreover, the test results by the 3D-CS method are in line with the already published reports of local waters.64-66 In a word, the 3D-CS method can be used for determine the concentration range of H2O2 in natural waters quickly and accurately, and the more the original coordinates are collected, the more accurate the measuring scale is, which fully demonstrates the potential application of 3D-CS method in rapid detection of H2O2.

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the H2O2. Based on this property, a 3D-CS was constructed by using the three factors (the initial velocities of the reaction, the maximum absorption peak values of the reaction and the colors of the reaction liquids) for rapid H2O2 detection in a large concentration range. This method is expected to be used for rapidly reading the concentration of the unknown H2O2 directly when enough coordinates of the known concentration of H2O2 were determined. It is applicable for not only the enzymatic reaction in accordance with Michaelis equation, but also the one that is inhibited by the too high concentration of H2O2. Furthermore, the developed method can be used to detect H2O2 in natural waters and displayed satisfactory results. The VOxQDs will be widely used in various fields such as environmental monitoring, biological analysis and so on. In addition, it is meaningful to study the multienzyme activities of VOxQDs and reveal the reaction processes and mechanisms for the design of multienzyme system for further widespread applications, such as anti-inflammatory, anti-tumor and maintenance of the metabolic balance.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem. xxxxx. Coordinates of varies H2O2 concentrations, HRTEM images of the VOxQDs, detection of •OH and O2- formation using FL measurement, catalytic oxidations of different concentrations of TMB in ethanol-BGS and PBS-BGS, linear relationship of the UV-Vis absorbance of the VOxQDs-TMB system against H2O2 concentration, assays of the triple-enzyme mimics of VOxQDs in PBS-BGS and different pH, the characterization of the formation of the elemental oxygen, •OH, O2-, oxygen vacancies of the VOxQDs; Table S1, Figure S1-S8.

AUTHOR INFORMATION Corresponding Author E-Mail: [email protected] (Y. H. Xu) Fax/Tel: +86-15264285082 Author Contributions

Figure 9. UV-Vis spectra and time-dependent absorbance changes of the VOxQDs-TMB system in the presence ofthree different natural waters: (A) Qingdao's spring rain, (B) the surface water of Dagu river, Qingdao and (C) aquariums water which contaminated with algae. (D) Colors of three reaction mixtures. (E) The specific coordinates of the three solutions in the threedimensional coordinate system. Data were presented as mean ± SD (n = 3)

CONCLUSIONS In summary, VOxQDs were synthesized by a simple one-pot ethanol-thermal method. Due to the different valence states in the as-prepared VOxQDs, they exhibited multifunctional enzyme mimic activities of OXD and POD in ethanol-BGS and of OXD, POD, and SOD in PBS. The VOxQDs had a unique property that can oxidate TMB to three different color oxidation products upon varying the concentration gradients of

The manuscript was written through contributions of all authors. /All authors have given approval to the final version of the manuscript. / ‡Lei Huang and Yusheng Niu contributed equally to this work.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21874079 and 21575071), Natural Science Foundation for Outstanding Young Scientists of Shandong Province (ZR2018JL011), Qingdao Science & Technology Planning Project (17-6-3-15-gx), and Science & Technology Fund Planning Project of Shandong Colleges and Universities (J16LA13 &J18KA112).

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VOx Quantum Dots with Multienzyme-Mimic Activities and the

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