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Jul 9, 2016 - Gen-Xia Cao, Xiu-Ming Wu, Yu-Ming Dong, Zai-Jun Li and Guang-Li Wang *. The Key Laboratory of Food Colloids and Biotechnology, Ministry ...
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In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase Gen-Xia Cao, Xiu-Ming Wu, Yu-Ming Dong, Zai-Jun Li and Guang-Li Wang * The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China; [email protected] (G.-X.C.); [email protected] (X.-M.W.); [email protected] (Y.-M.D.); [email protected] (Z.-J.L.) * Correspondence: [email protected]; Tel.: +86-510-85917090; Fax: +86-510-85917763 Academic Editors: Hui Wei and Derek J. Mcphee Received: 30 May 2016; Accepted: 7 July 2016; Published: 9 July 2016

Abstract: In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO4 3´ -capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-D-glucose to give 1-thio-β-D-gluconic acid which spontaneously hydrolyzes to β-D-gluconic acid and H2 S; the generated H2 S instantly reacts with Cd2+ in the presence of Na3 PO4 to give PO4 3´ -stabilized CdS QDs in situ. Under visible-light (λ ě 400 nm) stimulation, the PO4 3´ -capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h+ , ‚ OH, O2 ‚´ and a little H2 O2 , which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO4 3´ -capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 µg/L to 50 mg/L with a low detection limit of 6.6 µg/L. We believe the PO4 3´ -capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors. Keywords: glucose oxidase; photoswitchable oxidase mimetics; visible light; colorimetric sensor

1. Introduction Natural enzymes play an important role in biochemistry due to their high substrate specificity and high catalytic efficiency in catalyzing various meaningful reactions. Unfortunately, being a type of protein, natural enzymes suffer from some serious disadvantages: for example, (i) they can be easily denatured by environmental changes; (ii) they are prone to being digested by protease; and (iii) their preparation and purification are usually complex and expensive [1]. Accordingly, searching for artificial enzyme mimics with good stability and high catalytic capability is of great interest and urgently needed. Especially, the rapidly advancing field of nanotechnology supplies new possibilities for the development of enzyme mimics. Gao et al. [2] reported that Fe3 O4 nanoparticles (NPs) possessed an intrinsic peroxidase-like activity in 2007, which opened the door for developing various nanoscale materials such as enzyme mimetics in the biochemical field. Since then, many manufactured nanomaterials have been found to possess peroxidase-like activity including noble metals [3,4], metal oxides [5], carbon materials [6–9] and so on. Owning to the prominent advantages of low cost, high stability, ease of storage, and tunability in catalytic activity, these nanomaterial-based mimicking enzymes are promising candidates for natural enzymes in biological and biomedical applications [10–12]. However, almost all of these nanomaterial-based peroxidase mimetics are

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ready-made ones; to construct exquisite nanoenzyme that can be integrated with biomolecules is becoming a significant field. In this paper, we report an advanced method to detect the enzymatic activity of glucose oxidase (GOx) based on the GOx generation of S2´ anions followed by interaction with Cd2+ /Na3 PO4 to give PO4 3´ -capped CdS QDs in situ. Very interestingly, the PO4 3´ -capped CdS QDs were found to possess intrinsic oxidase-like activity under visible-light (λ ě 400 nm) stimulation, which could catalyze the oxidation of the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with dissolved oxygen acting as the electron acceptor. Based on the GOx-triggered in situ growth of PO4 3´ -capped CdS QDs with intrinsic enzyme-like activity, we offered a novel approach to detect the enzymatic activity of GOx with efficient signal amplification. The mechanism of the catalytic reaction of the photoswitchable oxidase mimetics was also detected, proving that the generation of photo-generated holes (h+ ), hydroxyl radicals (‚ OH), hydrogen peroxide (H2 O2 ) and especially superoxide anions (O2 ‚´ ) comprised the reactive species. Compared to natural horseradish peroxidase (HRP) or the widely studied peroxidase mimetics based on nanomaterials, the photoswitchable oxidase mimetics of PO4 3´ -capped CdS QDs displayed several distinct advantages, such as the avoidance of damaging hydrogen peroxide, excellent enzyme-like activity, good stability even in harsh environments, and the easily triggered/controlled activity by visible light irradiation, etc., which fully demonstrated its great application foreground in promising biosensing and biotechnology. Considering that GOx is one of the most common enzymes (highly specific for β-D-glucose) that has been widely used in analytical chemistry due to its high turnover, specificity and stability [13,14], we believe that the protocol of coupling GOx with photoswitchable oxidase mimetics may find wide applications in biosensors. 2. Results and Discussion 2.1. The Photoswitchable Oxidase Mimetics of PO4 3´ -Capped CdS QDs Generated by GOx-Mediated Biocatalysis GOx not only can enhance the oxidation of glucose, but it can also reinforce the oxidation of 1-thio-β-D-glucose to generate H2 S [15]. The generated H2 S could react immediately with cadmium cations and give CdS QDs. In addition, PO4 3´ was used as a stabilizer for the formed CdS QDs to prevent particle aggregation [16]. The size of the CdS QDs catalyzed by GOx is about 2–3 nm (Figure 1A) as confirmed high resolution transmission electron microscopy (TEM). From the UV/vis spectrum of the formed PO4 3´ -capped CdS QDs (Figure 1B), we observe an absorption band from 300 to 480 nm and a shoulder peak at about 380 nm. The presence of this shoulder peak is explained by the 1Sh –1Se excitonic transition [17] characteristic of CdS NPs with a diameter of ~2–3 nm from the work of Peng et al. [18], which confirmed the data of the TEM analysis. The emission spectrum indicated (blue line) a well-shaped peak at 550 nm with an excitation of 290 nm, also demonstrating the typical fluorescence characteristics of QDs. Using TMB as the typical substrate of peroxidase/oxidase [19], the enzyme-mimicking activity of CdS QDs or PO4 3´ -capped CdS QDs under visible-light irradiation was investigated. As indicated in Figure 2, the formed CdS QDs could hardly induce the oxidation of TMB in the absence of visible light (line a). When the visible light (λ ě 400 nm) was introduced, two apparent absorption peaks at 370 nm and 652 nm appeared for CdS QDs (line b) and PO4 3´ -capped CdS QDs (line c), which were characteristic absorption peaks of oxidized TMB (oxTMB). However, neither Cd2+ nor S2´ alone could catalyze the oxidation of TMB under visible light (λ ě 400 nm) irradiation (Figure S1), which demonstrated that the enzyme-like catalytic activity was due to the photo-triggered CdS QDs. The bare CdS without surface stabilizers was easy to gather, resulting in the decrease of catalytic effect. Therefore, we imported a series of stabilizers to prevent its aggregation (Figure S2A). As reported, thioglycolic acid (TGA) was usually used to stabilize QDs. Because the covalent binding of thiol (-SH) and Cd2+ occurred, a protective layer with negative charges was formed on the surface of the QDs. This protective layer with electrostatic repulsion prevents direct contact between the quantum dots,

2.1. The Photoswitchable Oxidase Mimetics of PO43−-Capped CdS QDs Generated by GOx-Mediated Biocatalysis GOx not only can enhance the oxidation of glucose, but it can also reinforce the oxidation of 1-thio-β-D-glucose to generate H2S [15]. The generated H2S could react immediately with cadmium Molecules 2016, 21, 902 3 of 10 cations and give CdS QDs. In addition, PO43− was used as a stabilizer for the formed CdS QDs to prevent particle Molecules 2016, 21, 902aggregation [16]. The size of the CdS QDs catalyzed by GOx is about 2–3 3 nm of 9 (Figure 1A) asformation confirmedofhigh resolution transmission electron microscopy (TEM). the UV/vis leading to the stable water-soluble nanoparticles. Similarly, Na3 PO dimethyl diallyl 4 , polyFrom DCdS -glucose. 43−-capped CdS were[16,20,21]. formed in the presence the enzymatic hydrolysis 1-thio-βspectrum of chloride the formed POof 43−-capped QDsPO (Figure 1B), weQDs observe an absorption band from ammonium (PDDA) and chitosan (CS) could also stabilize QDs However, TGA 2+ (1.5 mM), pH = 4.0. Dpeak -glucose (1 mM) and Cd of GOx (20 mg/L), 1-thio-β300 to 480 nm and a shoulder at about 380 nm. The presence of this shoulder peak is explained may inhibit the catalytic effect in our system because of its reducibility. PDDA, CS, Na3 PO4 could by the 1Sthe h–1S e excitonic transition [17] characteristic of CdS NPs witheffect a diameter of 4~2–3 improve catalytic effect with different degrees and the enhancement of Na3 PO wasnm the from most UsingofTMB as typical substrate of peroxidase/oxidase [19],analysis. the enzyme-mimicking activity the work PengS2A). etthe al.So [18], which confirmed the data of the TEM The emission spectrum obvious (Figure we chose Na PO as the stabilizer of CdS for obtaining enhanced catalytic 3 4 of CdS QDs or PO 43−-capped CdS QDs under visible-light irradiation was investigated. As indicated indicated (blue line) aS2B, well-shaped 550 nm with an excitation of 290 nm, off alsoindemonstrating activity. From Figure we foundpeak thatatthe catalytic oxidation process leveled 12 min. Thus, in Figure 2, the formed CdS QDs could hardly induce the oxidation of TMB in the absence of visible themin typical of QDs. 12 wasfluorescence chosen as thecharacteristics illumination time. light (line a). When the visible light (λ ≥ 400 nm) was introduced, two apparent absorption peaks at 3− 370 nm and 652 nm appeared for CdS QDs (line b) c), which were 0.15and PO4 -capped CdS QDs (line700 2+ characteristic absorption peaks of oxidized TMB (oxTMB). However, neither Cd600nor S2− alone could catalyze the oxidation of TMB under visible light (λ ≥ 400 nm) irradiation (Figure 500 S1), which 0.10 demonstrated that the enzyme-like catalytic activity was due to the photo-triggered400 CdS QDs. The bare CdS without surface stabilizers was easy to gather, resulting in the decrease of300 catalytic effect. Therefore, we imported a series of stabilizers to prevent its aggregation (Figure S2A). As reported, 0.05 200 thioglycolic acid (TGA) was usually used to stabilize QDs. Because the covalent binding of thiol 100 (-SH) and Cd2+ occurred, a protective layer with negative charges was formed on the surface of the 0 0.00 prevents direct contact between the quantum QDs. This protective layer with electrostatic repulsion 300 350 400 450 500 550 600 dots, leading to the formation of stable water-soluble nanoparticles. Similarly, Na3PO4, poly Wavelength(nm) dimethyl diallyl ammonium chloride (PDDA) and chitosan (CS) could also stabilize QDs [16,20,21]. FigureTGA 1. Characterization of the enzymatically CdS because QDs. (A) HRTEM image and PDDA, (B) the However, may inhibit the catalytic effect ingenerated our system its reducibility. Figure 1. Characterization of the enzymatically generated CdS QDs. (A)of HRTEM image and (B) the CS, 3−-capped CdS QDs produced by absorption (black line) and fluorescence (blue line) spectrum of PO 43´ Na3PO 4 could (black improve effect degrees and theCdS enhancement absorption line)the andcatalytic fluorescence (bluewith line) different spectrum of PO4 -capped QDs producedeffect by of Na3PO was the hydrolysis most obvious (Figure S2A). PO So4 3´ we-capped choseCdS Na3QDs PO4 were as the stabilizer of CdS for the4 enzymatic of 1-thio-βD -glucose. formed in the presence of GOxenhanced (20 mg/L),catalytic 1-thio-β-Dactivity. -glucose (1From mM) and Cd2+S2B, (1.5 mM), pH = 4.0. obtaining Figure we found that the catalytic oxidation process leveled off in 12 min. Thus, 12 min was chosen as the illumination time.

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Figure 2. 2. The (b)(b) CdS + TMB under visible-light irradiation; (c) Figure TheUV/Vis UV/Visspectra spectraofof(a)(a)CdS CdS+ TMB; + TMB; CdS + TMB under visible-light irradiation; 3− 3´ -capped -capped CdS QDs TMB+under irradiation. CdS QDsCdS were formed the presence PO4 PO (c) CdS+QDs TMB visible-light under visible-light irradiation. QDs wereinformed in the 4 2+ (1.5 mM), 2+ D -glucose (1 mM) and Cd pH = 4.0. Irradiation time: 12 min. of GOx (20 mg/L), 1-thio-βpresence of GOx (20 mg/L), 1-thio-β-D-glucose (1 mM) and Cd (1.5 mM), pH = 4.0. Irradiation time: Inset image is the corresponding color of the above solutions. 12 min. Inset image is the corresponding color of the above solutions.

Similar to natural enzymes, the relative catalytic activities of the PO43−-capped CdS QDs were Similar to natural enzymes, the relative catalytic activities of the PO4 3´ -capped CdS QDs were also influenced by the solution pH and temperature. It was found that the PO43−-capped CdS QDs also influenced by the solution pH and temperature. It was found that the PO4 3´ -capped CdS QDs could retain relatively high activity at a wide range of pH (Figure S2C) and temperature (Figure could retain relatively high activity at a wide range of pH (Figure S2C) and temperature (Figure S2D); S2D); the optimum reaction pH for PO43−-capped CdS QDs was 3.0, while HRP (using H2O2 as an the optimum reaction pH for PO4 3´ -capped CdS QDs was 3.0, while HRP (using H2 O2 as an electron electron acceptor) achieved the highest catalytic activity at a pH of 5.0, and lower or higher pH both acceptor) achieved the highest catalytic activity at a pH of 5.0, and lower or higher pH both largely largely inhibited the catalytic activity. From Figure S2D, we can see that the PO43−-capped CdS QDs inhibited the catalytic activity. From Figure S2D, we can see that the PO4 3´ -capped CdS QDs keep a keep a high catalytic activity in a wide range of temperatures from 20 to 90 °C, and the optimum high catalytic activity in a wide range of temperatures from 20 to 90 ˝ C, and the optimum reaction reaction temperature was 43 °C, while HRP reached its maximum catalytic activity around 35 °C temperature was 43 ˝ C, while HRP reached its maximum catalytic activity around 35 ˝ C and showed a and showed a significant decrease in catalytic activity at lower or higher temperatures. These significant decrease in catalytic3−activity at lower or higher temperatures. These results demonstrated results demonstrated that PO4 -capped CdS QDs as enzyme mimetics showed better stability and that PO4 3´ -capped CdS QDs as enzyme mimetics showed better stability and higher activity even higher activity even under harsher conditions than that of HRP (using H2O2 as an electron under harsher conditions than that of HRP (using H2 O2 as an electron acceptor). acceptor). We further examined the enzyme-like catalytic properties of PO43−-capped CdS QDs under visible-light irradiation by steady-state kinetics. As shown in Figure S3, under the optimal

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We further examined the enzyme-like catalytic properties of PO4 3´ -capped CdS QDs under visible-light irradiation by steady-statecurves kinetics. As obtained shown in for Figure S3, under the conditions, conditions, typical Michaelis–Menten were PO43− -capped CdSoptimal QDs with TMB as 3 ´ typical Michaelis–Menten curves were obtained for -capped CdS constant QDs with(KTMB asindicator a substrate 4 a substrate at a certain range of concentrations. ThePO Michaelis-Menten m), an of at a certain range of concentrations. The Michaelis-Menten constant (K ), an indicator of an enzyme’s m an enzyme’s affinity for its substrate, was obtained using Lineweaver–Burk plots (Figure S3 and affinity for its obtained usingwere Lineweaver–Burk (Figure υS3= and The Figure S4). Thesubstrate, apparentwas kinetic parameters calculated by plots the equation VmaxFigure × [S]/(KS4). m + [S]), apparent kinetic parameters werereaction calculated by[S] theisequation υ = V max ˆ is m + [S]), where where υ is the initial enzymatic rate, the concentration of[S]/(K the substrate, Vmax is υthe the initial enzymatic reaction rate, [S] is the concentration of the substrate, V is the maximum maxapparent Km value maximum enzymatic reaction rate and Km is the Michaelis-Menten constant. The enzymatic reaction ratePO and Km is the Michaelis-Menten The apparent of was the m value of the photo-activated 43−-capped CdS QDs with TMB constant. as a substrate was 97.7 K μM, which 3´ -capped CdS QDs with TMB as a substrate was 97.7 µM, which was much lower photo-activated PO −1 4 much lower than that of HRP (Km = 434 μM). In addition, the Vmax was 42.86 nM·s . These results than that ofthat HRP = 434 µM). addition, the Vkind 42.86 nM¨ s´1 . These indicated m-capped max was indicated PO(K 43− CdSInQDs as a new of mimetic enzyme had results a higher affinitythat for 3 ´ PO -capped CdS QDs as a new kind of mimetic enzyme had a higher affinity for TMB than did HRP. 4 than TMB did HRP. 3´ -Capped CdS QDs 2.2. Mechanism of 2.2. Mechanism of Photoswitchable Photoswitchable Enzyme-Like Enzyme-Like Activity Activity of of PO PO443−-Capped CdS QDs

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With of verifying the catalytic mechanism, we primarily bubbledbubbled high-purity nitrogen With the thepurpose purpose of verifying the catalytic mechanism, we primarily high-purity into the catalytic reaction system for 20 min. From Figure S5, we could see the absorbance peak of nitrogen into the catalytic reaction system for 20 min. From Figure S5, we could see the absorbance oxTMB distinctly, which demonstrated that dissolved oxygen as an oxidant an integral peak of declined oxTMB declined distinctly, which demonstrated that dissolved oxygen as an was oxidant was an part of the catalytic oxidation system. As a result, we called the CdS QDs with photoswitchable integral part of the catalytic oxidation system. As a result, we called the CdS QDs with enzyme-like activity photoswitchable mimetics.oxidase mimetics. photoswitchable enzyme-like activity oxidase photoswitchable For system responsible forfor thethe catalyzed oxidization, we For further furtherclarifying clarifyingthe theactive activespecies speciesofofthe the system responsible catalyzed oxidization, applied a train of scavengers to capture the active species. As we know, KI and EDTA are scavengers of we applied a train of scavengers to capture the active species. As we know, KI and EDTA are + ) [22], NaHCO and t-butanol are scavengers of hydroxyl radicals (‚ OH) [23], photo-generated holes (h scavengers of photo-generated holes (h3+) [22], NaHCO3 and t-butanol are scavengers of hydroxyl ‚´ )[24] and catalase (CAT) can superoxide dismutase (SOD) is the scavenger of superoxide anionsof(Osuperoxide 2 radicals (•OH) [23], superoxide dismutase (SOD) is the scavenger anions (O2•−)[24] and catalyze the decomposition of H O into water and oxygen [25]. As indicated in when these 2 catalase (CAT) can catalyze the 2decomposition of H2O2 into water and oxygen Figure [25]. As3,indicated in scavengers were these introduced in our system, respectively, of oxTMB declined in varying Figure 3, when scavengers were introduced in the ourabsorbance system, respectively, the absorbance of degrees. These experiments proved These that the h+ , ‚ OH, H especially O2 ‚´ the reactive 2 O2 and oxTMB declined in varying degrees. experiments proved that the h+, •OH, H2were O2 and especially species responsible for TMB oxidation. •− O2 were the reactive species responsible for TMB oxidation.

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Figure 3. 3. The The effects effectsof ofdifferent differentscavengers scavengersononthe theoxidation oxidation TMB under visible-light irradiation Figure of of TMB under visible-light irradiation by 3´ 43−-capped CdS QDs. by PO PO4 -capped CdS QDs.

We introduced photoelectrochemistry and electrochemistry experiments to confirm the reactive We introduced photoelectrochemistry and electrochemistry experiments to confirm the reactive species generation mechanism of illuminated CdS QDs. Firstly, we investigated the photocurrent of species generation mechanism of illuminated CdS QDs. Firstly, we investigated the photocurrent PO43−-capped CdS QDs in phosphate buffer. From Figure S6, we can see that the PO43−-capped CdS of PO4 3´ -capped CdS QDs in phosphate buffer. From Figure S6, we can see that the PO4 3´ -capped QDs promptly generate a stable photocurrent with a reproducible response to on/off cycles, CdS QDs promptly generate a stable photocurrent with a reproducible response to on/off cycles, demonstrating the effective electron/hole generation and transfer of photoactivated PO34´3−-capped demonstrating the effective electron/hole generation and transfer of photoactivated PO4 -capped CdS QDs. Subsequently, we employed linear sweep voltammetry (LSV) to study the conduction CdS QDs. Subsequently, we employed linear sweep voltammetry (LSV) 3− to study the conduction band (CB) (Figure S7A) and valence band (VB) (Figure S7B) edge of PO34´ -capped CdS QDs. The band (CB) (Figure S7A) and valence band (VB) (Figure S7B) edge of PO4 -capped CdS QDs. The results showed that PO43−-capped CdS QDs had a CB edge at −0.77 V and a VB edge at 0.93 V vs. results showed that PO4 3´ -capped CdS QDs had a CB edge at ´0.77 V and a VB edge at 0.93 V Ag/AgCl (saturated KCl). That meant the CB and VB potentials of PO43−-capped3CdS QDs were −0.57 vs. Ag/AgCl (saturated KCl). That meant the CB and VB potentials of PO4 ´ -capped CdS QDs and 1.13 V vs. normal hydrogen electrodes (NHE), respectively. CdS is a favorable semiconductor material; under visible-light illumination, the electron of VB was excited into the CB and led to the VB producing h+. Because of the potential gradient between the CB (−0.57 V vs. NHE) of CdS QDs and the reduction potential of oxygen (0.815 V vs. NHE) [20], the oxygen in aqueous solution could

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were ´0.57 and 1.13 V vs. normal hydrogen electrodes (NHE), respectively. CdS is a favorable semiconductor material; under visible-light illumination, the electron of VB was excited into the Molecules 2016, 21, 902 5 of 9 CB and2016, led 21, to 902 the VB producing h+ . Because of the potential gradient between the CB (´0.57 5V Molecules ofvs. 9 NHE) of CdS QDs and the reduction potential •−of oxygen (0.815 V vs. NHE) [20], the oxygen in aqueous accept the excited electrons and formed O2•− or ••OH. The h+‚+ were able+ to oxidize TMB directly ‚´ orh accept the excited electrons andelectrons formed and O2 formed or OH. wereThe able oxidize directly solution could accept the excited O2The OH. h to were able toTMB oxidize TMB because the VB potential of CdS QDs was above that of TMB with an oxidation potential in the range because VB potential of CdS QDs was QDs abovewas thatabove of TMB with an oxidation the range directly the because the VB potential of CdS that of TMB with+ an potential oxidationin potential in of 0.22–0.7 V [26]. The above experiments proved that O2•−•−, ••OH and h+ were the main reactive ‚´ , ‚h of [26]. The experiments proved that O2 ,that OHO2and were main the reactive the0.22–0.7 range ofV0.22–0.7 V above [26]. The above experiments proved OH andthe h+ were main species responsible for TMB oxidation (Scheme 1). species for TMB for oxidation (Scheme (Scheme 1). reactiveresponsible species responsible TMB oxidation 1).

Scheme 1. The mechanism of the photo-activated oxidase-like activity of PO43−-capped CdS QDs. Scheme 1. The mechanism of the photo-activated oxidase-like activity of PO43− -capped CdS QDs. Scheme 1. The mechanism of the photo-activated oxidase-like activity of PO4 3´ -capped CdS QDs.

2.3. Probing the Activity of Glucose Oxidase Using PO43−-Capped CdS QDs 2.3. Probing the Activity of Glucose Oxidase Using PO43−3-Capped CdS QDs 2.3. Probing the Activity of Glucose Oxidase Using PO4 ´ -Capped CdS QDs The coupling of biomolecules, for example natural enzymes, with nanomaterials formed in situ The coupling of biomolecules, for example natural enzymes, with nanomaterials formed in situ for target analytesofhas high sensitivity due to the low background signals, andformed it has in received The coupling biomolecules, for example natural enzymes, with nanomaterials situ for for target analytes has high sensitivity due to the low background signals, and it has received increasing attention by researchers [27,28]. Aslow a biocatalyst, natural enzymes playreceived an important role target analytes has high sensitivity due to the background signals, and it has increasing increasing attention by researchers [27,28]. As a biocatalyst, natural enzymes play an important role in the catalytic reaction, and even a very tiny amount of the enzyme can stimulate the occurrence of attention by researchers [27,28]. As a biocatalyst, natural enzymes play an important role in the in the catalytic reaction, and3− even a very tiny amount of the enzyme can stimulate the occurrence of the catalytic reaction. 4 -capped CdSamount QDs formed in situ by biocatalysis is an excellent catalytic reaction, and PO even a very tiny of the enzyme canGOx stimulate the occurrence of the the catalytic reaction. 3PO 43−-capped CdS QDs formed in situ by GOx biocatalysis is an excellent ´ -capped mimetic rapidly; it catalyzes the oxidation of TMB andmimetic further catalytic oxidase reaction.that PO4forms CdSonce QDs produced, formed in situ by GOx biocatalysis is an excellent mimetic oxidase that forms rapidly; once produced, it catalyzes the oxidation of TMB and further produces anforms amplified Further, itour detection does notofrequire the use produces of intricate oxidase that rapidly;signal. once produced, catalyzes the oxidation TMB and further an produces an amplified signal. Further, our detection does not require the use of intricate instruments, which makes it detection cheaper and easy tothe operate. Based on the catalytic growth of amplified signal. Further, our doesmore not require use of intricate instruments, which makes instruments, which makes it cheaper and more easy to operate. Based on the catalytic growth of 3´ -capped 3−-capped CdS QDs, we PO 43−-capped CdS QDs in situ and the intrinsic oxidase-like property of PO 4 it cheaper and more easy to operate. Based on the catalytic growth of PO CdS QDs in situ 4 PO43−-capped CdS QDs in situ and the intrinsic oxidase-like property of PO 43−-capped CdS QDs, we developed a facile colorimetric method amplification to detect thecolorimetric enzymatic and the intrinsic oxidase-like property of with PO4 3´efficient -cappedsignal CdS QDs, we developed a facile developed a facile colorimetric method with efficient signal amplification to detect the enzymatic activity GOx (Scheme 2). amplification to detect the enzymatic activity of GOx (Scheme 2). method of with efficient signal activity of GOx (Scheme 2).

Scheme 2. Detection of GOx activity through forming the photoswitchable oxidase mimetics of Scheme 2. 2. Detection DetectionofofGOx GOx activity activity through through forming forming the the photoswitchable photoswitchable oxidase oxidase mimetics mimetics of of Scheme PO43−3−3´ -capped CdS QDs with the enzymatic product of GOx. PO -capped CdS QDs with the enzymatic product of GOx. PO44 -capped CdS QDs with the enzymatic product of GOx.

For the sake of acquiring a better catalytic effect, we adjusted and controlled the reaction For the sake of acquiring a better catalytic effect, we adjusted and controlled the reaction conditions of the GOx catalytic reaction, such as the reaction time and the concentration of conditions of the GOx catalytic reaction, such as the reaction time and the concentration of 1-thio-β-D-glucose. As shown in Figure S8A, the enzyme catalytic reaction proceeded quickly and 1-thio-β-D-glucose. As shown in Figure S8A, the enzyme catalytic reaction proceeded quickly and achieved a balance in 60 min. As can be seen from Figure S8B, we can see that with the increase of achieved a balance in 60 min. As can be seen from Figure S8B, we can see that with the increase of the 1-thio-β-D-glucose concentration, the absorbance of oxTMB gradually enhances, meaning that the 1-thio-β-D-glucose concentration, the absorbance of oxTMB gradually enhances, meaning that

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For the sake of acquiring a better catalytic effect, we adjusted and controlled the reaction conditions of the GOx catalytic reaction, such as the reaction time and the concentration of 1-thio-β-D-glucose. As shown in Figure S8A, the enzyme catalytic reaction proceeded quickly and achieved a balance in 60 min. As can be seen from Figure S8B, we can see that with the increase of the 1-thio-β-D-glucose Molecules 2016, 21, 902 6 of 9 concentration, the absorbance of oxTMB gradually enhances, meaning that the catalytic effect is strengthened step by step. When step the 1-thio-βD -glucose concentration is greater than 1 mM, the the catalytic effect is strengthened by step. When the 1-thio-βD-glucose concentration is greater absorbance of oxTMB hardly increases because of the depletion of GOx. Thus, we chose 60 min as than 1 mM, the absorbance of oxTMB hardly increases because of the depletion of GOx. Thus,the we enzyme timeenzyme and 1 mM as thetime concentration D -glucose. of thio-β-D-glucose. chose 60reaction min as the reaction and 1 mM of as thio-βthe concentration To Toconfirm confirmthat thatthe theactivity activityofofGOx GOxwas wasthe themain mainfactor factorininthe theproduction productionof ofCdS CdSQDs, QDs, we we verified the influence of the natural substrate βD -glucose. Theoretically, the artificial substrate verified the influence of the natural substrate β-D-glucose. Theoretically, the artificial substrate and and natural were supposed competefor forbinding bindingwith withthe the active active site site of the the natural oneone were supposed to to compete of GOx. GOx. Thus, Thus, the the presence of βD -glucose consumes a part of the GOx and leads to the diminution of GOx to presence of β-D-glucose consumes a part of the GOx and leads to the diminution of GOx tocatalyze catalyze 1-thio-β1-thio-β-DD-glucose. -glucose. When When the the concentration concentration of of1-thio-β1-thio-β-DD-glucose -glucose is is fixed, fixed, the theincreasing increasingamount amountof of βD -glucose leads to a decreasing amount of its oxidative decomposition to H S and gluconic acid and 2 β-D-glucose leads to a decreasing amount of its oxidative decomposition to H2S and gluconic acid ´ -capped CdS QDs (Figure S8C). Based on the above consequently weakens the catalytic effect of PO4 3of and consequently weakens the catalytic effect PO43−-capped CdS QDs (Figure S8C). Based on the 3 ´ results, we can see of PO4 -capped QDs CdS in situ originated from the enzymatic above results, wethat can the seeformation that the formation of PO43−CdS -capped QDs in situ originated from the reaction of 1-thio-βD -glucose. enzymatic reaction of 1-thio-β-D-glucose. To Tostudy studythe thesensitivity sensitivityof ofthe theproposed proposed method method for for the the detection detection of of GOx GOx activity, activity, different different amounts of commercially available GOx were added to the system and the absorption spectrum amounts of commercially available GOx were added to the system and the absorption spectrumof of oxTMB oxTMBwas wasrecorded. recorded. As As illustrated illustrated in in Figure Figure 4A, 4A, with withthe theincrease increaseof ofthe theGOx GOxconcentration, concentration,the the absorbance absorbanceof ofoxTMB oxTMBatat652 652nm nmincreased increasedgradually graduallyand andthe theabsorbance absorbanceincreased increasedlinearly linearlywith withthe the logarithmic enzymatic activity of GOx over the range from 25 µg/L to 50 mg/L with a detection limit logarithmic enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a detection limit of 3)(Figure (Figure 4B). 4B). The The detection detection limit limit of of this of6.6 6.6µg/L μg/L (S/N (S/N ==3) this method method for for the the enzymatic enzymatic activity activityof ofGOx GOx was wascomparable comparableor oreven evenlower lowerthan thanthat thatof ofother othermethods methods[15,29] [15,29]reported, reported,and anditithas hasaasufficiently sufficiently wider to other other methods methods[30]. [30].This Thiscolorimetric colorimetric method detection of widerlinear linear range range compared compared to method forfor thethe detection of the the enzymatic activity of GOx is convenient for detection with the naked eye and fairly inexpensive enzymatic activity of GOx is convenient for detection with the naked eye and fairly inexpensive compared comparedtotoother othermethods methodswhich whichare aretime-consuming time-consumingororrequire requirespecialized specializedinstruments. instruments.

A

B 0.8

50 mg/L

0.025 mg/L

0.2

0.8

0.6

Abs(a.u.)

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Abs(a.u.)

Abs(a.u.)

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0.6 0.4 0.2 0.0 0.1

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800

1

10

Log[GOx](mg/L)

0.0 0

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[GOx](mg/L)

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Figure4.4.Analytical Analyticalperformances performancesofofthe theprotocol protocolfor forprobing probingthe theactivity activityofofGOx. GOx.(A) (A)UV/vis UV/visspectra spectra Figure 3− ofoxTMB oxTMBcatalyzed catalyzedby bythe thePO PO3´ 4 -capped CdS QDs formed in situ under visible-light irradiation in of -capped CdS QDs formed in situ under visible-light irradiation in 4 the presence of different concentrations ofGOx. GOx.From Frombottom bottomto totop, top,the theconcentrations concentrationsof ofGOx GOxare are the presence of different concentrations of 0.025, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 10.0, 20.0, 50.0 mg/L. The inset shows the corresponding color change 0.025, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 10.0, 20.0, 50.0 mg/L. The inset shows the corresponding color change of of oxTMB; Absorption change linear relationship (insert curve) between absorbance oxTMB; (B) (B) Absorption change andand the the linear relationship (insert curve) between the the absorbance of of oxTMB 652and nmtheand the concentration of GOx. error bars indicate relative standard oxTMB at 652atnm concentration of GOx. The error The bars indicate relative standard deviation of deviation of four repeated experiments. four repeated experiments.

3. Materials and Methods 3.1. Chemicals and Materials The 1-thio-β-D-glucose was purchased from J&K (Shanghai, China). Glucose oxidase type VII (GOx), horseradish peroxidase (HRP), superoxide dismutase (SOD) from bovine liver, catalase (CAT), poly dimethyl diallyl ammonium chloride (PDDA, 20%, w/w in water, molecular weight = 200,000–350,000) and were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Na2S·9H2O was purchased from Shanghai Tongya Chemical Technology Co., Ltd (Shanghai, China). Glucose,

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3. Materials and Methods 3.1. Chemicals and Materials The 1-thio-β-D-glucose was purchased from J&K (Shanghai, China). Glucose oxidase type VII (GOx), horseradish peroxidase (HRP), superoxide dismutase (SOD) from bovine liver, catalase (CAT), poly dimethyl diallyl ammonium chloride (PDDA, 20%, w/w in water, molecular weight = 200,000–350,000) and were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Na2 S¨ 9H2 O was purchased from Shanghai Tongya Chemical Technology Co., Ltd (Shanghai, China). Glucose, ethylene diamine tetraacetic acid (EDTA), KI, t-butanol, 3,31 ,5,51 -tetramethylbenzidine (TMB), 3CdSO4 ¨ 8H2 O, thioglycolic acid (TGA), chitosan (CS), Na3 PO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade. All solutions were prepared with ultrapure water (18.2 MΩ¨ cm´1 ) obtained from a Healforce water purification system. 3.2. Instrumentation High resolution transmission electron microscopy (HRTEM) images of PO4 3´ -capped CdS QDs were obtained on a JEOL JEM-2100 transmission electron microscope (Hitachi, Japan). The fluorescence spectra analysis and the resonance light scattering spectra were carried out on a Varian Cary Eclipse fluorescence spectrophotometer at room temperature. UV/Vis absorption spectroscopic measurements were carried out using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., Beijing, China). A 300 W Xe lamp (NBeT, Beijing, China) equipped with an ultraviolet cutoff filter (λ ě 400 nm) was used as the irradiation source. Photoelectrochemical measurements were performed with a homemade photoelectrochemical system. Photocurrent was measured on a CHI 800C electrochemical workstation (Shanghai, China). PO4 3´ -capped CdS QDs modified ITO electrode was employed as the working electrode. A Pt wire was used as the counter electrode and a saturated Ag/AgCl as the reference electrode. All the photocurrent measurements were performed at a constant potential of 0 V (vs. saturated Ag/AgCl) in 0.2 M Na2 SO4 solution as the supporting electrolyte. Linear sweep voltammetry (LSV) was used to determine the conduction/valence band edge of the PO4 3´ -capped CdS QDs, which was performed with a CHI 800C electrochemical workstation at room temperature under N2 atmosphere. A conventional three-electrode cell was used, including a Pt wire counter electrode, a saturated Ag/AgCl reference electrode. A glassy carbon electrode was used as working electrode. A 0.2 M Na2 SO4 solution containing PO4 3´ -capped CdS QDs was used as the electrolyte solution. The detection of GOx activity by the absorption method was proceeded on 96 well plates by the microplate reader (SpectraMax M5, Sunnyvale, California, USA). 3.3. The Colorimetric Detection of Glucose Oxidase Activity A certain amount of 1-thio-β-D-glucose were incubated with different amounts of GOx in citrate buffer (10 mM, pH 4.0) for 60 min at 37 ˝ C. After that, 20 µL Cd2+ /Na3 PO4 mixture was added to the samples. Subsequently, the mixed solution was added with 20 µL of 5 mM TMB and then diluted to 200 µL by acetate buffer (200 mM, pH 4.0) and illuminated under visible light irradiation (λ ě 400 nm) for 10 min to allow development of the blue color, and the absorbance of the oxidized TMB (oxTMB) at 652 nm was measured. 4. Conclusions In this work, we introduced a new, amplified approach for probing the activity of GOx based on coupling the enzymatic reaction of GOx and its in situ generation of PO4 3´ -capped CdS QDs with photoswitchable oxidase-mimicking activity. Under visible-light (λ ě 400 nm) stimulation, the generated PO4 3´ -capped CdS QDs were found to possess intrinsic oxidase-like activity which could catalyze the oxidation of the typical substrate (TMB) with dissolved oxygen acting as the electron acceptor. Kinetic analysis proved that the catalysis was in accordance with the typical Michaelis–Menten kinetics and had a higher affinity for TMB than that of HRP. The catalytic mechanism

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investigations indicated that photo-generated holes (h+ ), hydroxyl radicals (‚ OH), and especially superoxide anions (O2 ‚´ ) were the reactive species for the catalytic reaction. By taking advantage of the GOx-triggered growth of PO4 3´ -capped CdS in situ and its intrinsic oxidase-like property, a facile, sensitive and selective colorimetric method was developed to probe the activity of GOx. It is expected that the PO4 3´ -capped CdS QDs generated in situ by GOx with photo-stimulated enzyme-mimicking activity may find wide potential applications in the fields of catalysis, biochemistry and biotechnology. Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/21/7/902/s1. Acknowledgments: This work was supported by The National Natural Science Foundation of China (21575052, 21275065), and the Fundamental Research Funds for the Central Universities (JUSRP51314B) and the Opening Foundation of the State Key Laboratory of Analytical Chemistry for Life Science of Nanjing University (KLACLS1008). Author Contributions: Gen-Xia Cao, Guang-Li Wang conceived and designed the experiments; Gen-Xia Cao performed the experiments and analyzed the data; Yu-Ming Dong, Xiu-Ming Wu, and Zai-Jun Li contributed reagents/materials/analysis tools; Gen-Xia Cao and Guang-Li Wang wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript: GOx CdS QDs TMB NPs h+ ‚ OH O2 ‚´ HRP SOD CAT PDDA TGA CS

glucose oxidase cadmium sulfide quantum dots 3,31 ,5,51 -tetramethylbenzydine Nanoparticles photo-generated holes hydroxyl radical superoxide anion horseradish peroxidase superoxide dismutase Catalase poly dimethyl diallyl ammonium chloride thioglycolic acid Chitosan

References 1. 2.

3. 4.

5. 6.

7.

8.

Wei, H.; Wang, E. Fe3 O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2 O2 and glucose detection. Anal. Chem. 2008, 80, 2250–2254. [CrossRef] [PubMed] Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577–583. [CrossRef] [PubMed] Jv, Y.; Li, B.X.; Cao, R. Positively-charged gold nanoparticles as peroxidiase mimic and their application in hydrogen peroxide and glucose detection. Chem. Commun. 2010, 46, 8017–8019. [CrossRef] [PubMed] He, W.; Wu, X.; Liu, J.; Hu, X.; Zhang, K.; Hou, S.; Xie, S. Design of AgM bimetallic alloy nanostructures (M = Au, Pd, Pt) with tunable morphology and peroxidase-like activity. Chem. Mater. 2010, 22, 2988–2994. [CrossRef] Mu, J.S.; Wang, Y.; Zhao, M.; Zhang, L. Intrinsic peroxidase-like activity and catalase-like activity of Co3 O4 nanoparticles. Chem. Commun. 2012, 48, 2540–2542. [CrossRef] [PubMed] André, R.; Natálio, F.; Humanes, M.; Leppin, J.; Heinze, K.; Wever, R.; Schröder, H.C.; Müller, W.E.G.; Tremel, W. V2 O5 nanowires with an intrinsic peroxidase-like activity. Adv. Funct. Mater. 2011, 21, 501–509. [CrossRef] Shi, W.B.; Wang, Q.L.; Long, Y.J.; Cheng, Z.L.; Chen, S.H.; Zheng, H.Z. Carbon nanodots as peroxidase mimetics and their applications to glucose detection. Chem. Commun. 2011, 47, 6695–6697. [CrossRef] [PubMed] Song, Y.; Wang, X.; Zhao, C.; Qu, K.; Ren, J.; Qu, X. Label-free colorimetric detection of single nucleotide polymorphism by using single-walled carbon nanotube intrinsic peroxidase-like activity. Chem. Eur. J. 2010, 16, 3617–3621. [CrossRef] [PubMed]

Molecules 2016, 21, 902

9. 10. 11. 12.

13.

14.

15. 16.

17. 18. 19.

20.

21.

22. 23.

24. 25.

26.

27.

28.

9 of 10

Cui, R.J.; Han, Z.D.; Zhu, J.J. Helical carbon nanotubes: Intrinsic peroxidase catalytic activity and its application for biocatalysis and biosensing. Chem. Eur. J. 2011, 17, 9377–9384. [CrossRef] [PubMed] Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J.M. Oxidase-like activity of polymer-coated cerium oxide nanoparticles. Angew. Chem. Int. Ed. 2009, 48, 2308–2312. [CrossRef] [PubMed] Wang, X.X.; Wu, Q.; Shan, Z.; Huang, X.M. BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection. Biosen. Bioelectron. 2011, 26, 3614–3619. [CrossRef] [PubMed] Wang, G.L.; Jin, L.Y.; Dong, Y.M.; Wu, X.M.; Li, Z.J. Intrinsic enzyme mimicking activity of gold nanoclusters upon visible light triggering and its application for colorimetric trypsin detection. Biosens. Bioelectron. 2015, 64, 523–529. [CrossRef] [PubMed] Gerritsen, M.; Kros, A.; Lutterman, J.; Nolte, R.; Jansen, J. A percutaneous device as model to study the in vivo performance of implantable amperometric glucose sensors. J. Mater. Sci. Mater. Med. 2001, 12, 129–134. [CrossRef] [PubMed] Karmali, K.; Karmali, A.; Teixeira, A.; Marcelo Curtob, M.J. Assay for glucose oxidase from aspergillus niger and penicillium amagasakiense by fourier transform infrared spectroscopy. Anal. Biochem. 2004, 333, 320–327. [CrossRef] [PubMed] Saa, L.; Pavlov, V. Enzymatic growth of quantum dots: Applications to probe glucose oxidase and horseradish peroxidase and sense glucose. Small 2012, 8, 3449–3455. [CrossRef] [PubMed] Malashikhina, N.; Garai-Ibabe, G.; Pavlov, V. Unconventional application of conventional enzymatic substrate: First fluorogenic immunoassay based on enzymatic formation of quantum dots. Anal. Chem. 2013, 85, 6866–6870. [CrossRef] [PubMed] Matsumoto, H.; Sakata, T.; Mori, H.; Yoneyama, H. Preparation of Monodisperse CdS Nanocrystals by size selective photocorrosion. J. Phys. Chem. 1996, 100, 13781–13785. [CrossRef] Yu, W.W.; Qu, L.H.; Guo, W.Z.; Peng, X.G. Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem. Mater. 2003, 15, 2854–2860. [CrossRef] Jin, L.Y.; Dong, Y.M.; Wu, X.M.; Cao, G.X.; Wang, G.L. Versatile and amplified biosensing through enzymatic cascade reaction by coupling alkaline phosphatase in situ generation of photoresponsive nanozyme. Anal. Chem. 2015, 87, 10429–10436. [CrossRef] [PubMed] Wang, G.L.; Liu, K.L.; Shu, J.X.; Gu, T.T.; Wu, X.M.; Dong, Y.M.; Li, Z.J. A novel photoelectrochemical sensor based on photocathode of PbS quantum dots utilizing catalase mimetics of bio-bar-coded platinum nanoparticles/G-quadruplex/hemin for signal amplification. Biosen. Bioelectron. 2015, 69, 106–112. [CrossRef] [PubMed] Wang, G.L.; Xu, X.F.; Qiu, L.; Dong, Y.M.; Li, Z.J.; Zhang, C. Dual responsive enzyme mimicking activity of AgX (X = Cl, Br, I) nanoparticles and its application for cancer cell detection. ACS Appl. Mater. Interfaces 2014, 6, 6434–6442. [CrossRef] [PubMed] Cao, J.; Xu, B.; Luo, B.; Lin, H.; Chen, S. Preparation, characterization and visible-light photocatalytic activity of AgI/AgCl/TiO2 . Appl. Surf. Sci. 2011, 257, 7083–7089. [CrossRef] An, C.; Wang, J.; Qin, C.; Jiang, W.; Wang, S.; Li, Y.; Zhang, Q. Synthesis of Ag@AgBr/AgCl heterostructured nanocashews with enhanced photocatalytic performance via anion exchange. J. Mater. Chem. 2012, 22, 13153–13158. [CrossRef] Wang, W.; Jiang, X.; Chen, K. Iron phosphate microflowers as peroxidase mimic and superoxide dismutase mimic for biocatalysis and biosensing. Chem. Commun. 2012, 48, 7289–7291. [CrossRef] [PubMed] Wu, T.; Liu, G.; Zhao, J.; Hidaka, H.; Serpone, N. Evidence for H2 O2 generation during the TiO2 -assisted photodegradation of dyes in aqueous dispersions under visible light illumination. J. Phys. Chem. B 1999, 103, 4862–4867. [CrossRef] Marquez, L.A.; Dunford, H.B. Mechanism of the oxidation of 3,5,31 ,51 -tetramethylbenzidine by myeloperoxidase determined by transient-and steady-state kinetics. Biochemistry 1997, 36, 9349–9355. [CrossRef] [PubMed] Zhang, Y.Y.; Zhou, H.; Wu, P.; Zhang, H.R.; Xu, J.J.; Chen, H.Y. In situ activation of CdS electrochemiluminescence film and its application in H2 S detection. Anal. Chem. 2014, 86, 8657–8664. [CrossRef] [PubMed] Liu, S.; Wang, X.; Pang, S.; Na, W.D.; Yan, X.; Su, X.G. Fluorescence detection of adenosine-51 -triphosphate and alkaline phosphatase based on the generation of CdS quantum dots. Anal. Chim. Acta 2014, 827, 103–110. [CrossRef] [PubMed]

Molecules 2016, 21, 902

29.

30.

10 of 10

Okuma, H.; Sekimukai, S.; Hoshi, M.; Toyama, K.; Watanabe, E. Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities. Enzyme Microb. Technol. 1989, 11, 824–829. [CrossRef] Wu, M.; Lin, Z.; Schäferling, M.; Dürkop, A.; Wolfbeis, O.S. Fluorescence imaging of the activity of glucose oxidase using a hydrogen-peroxide-sensitive europium probe. Anal. Biochem. 2005, 340, 66–73. [CrossRef] [PubMed]

Sample Availability: Samples of the compounds are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).