Co3O4âAu Polyhedra: A Multifunctional Signal Amplifier for Sensitive

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Co3O4-Au Polyhedra: A New Multifunctional Signal Amplifier for Sensitive Photoelectrochemical Assay Ruiying Yang, Kang Zou, Yanmei Li, Leixia Meng, Xiaohua Zhang, and Jinhua Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02134 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Analytical Chemistry

Co3O4-Au Polyhedra: A New Multifunctional Signal Amplifier for Sensitive Photoelectrochemical Assay Ruiying Yang, Kang Zou, Yanmei Li, Leixia Meng, Xiaohua Zhang, Jinhua Chen∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China

∗

Corresponding author. Tel.: +86-731-88821848 E-mail address: [email protected] 1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Taking zeolitic imidazolate framework (ZIF-67) as the precursor, p-type semiconducting Co3O4-Au polyhedra were synthesized and used as the signal amplifier to construct a sensitive photoelectrochemical (PEC) sensor for caspase-3 activity assay. Here, the n-type semiconductor Bi2S3-modified indium−tin oxide (ITO) slice

was

used

as

the

photoelectrode.

After

immobilization

of

the

biotin-DEVD-peptide (biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Cys) onto the Bi2S3 surface, the streptavidin-labeled Co3O4-Au polyhedra were introduced to the sensing platform via the specific interaction between biotin and streptavidin. The Co3O4-Au polyhedra can not only quench the photocurrent of the Bi2S3 because of the competitive consumption of electron donors and exciting light energy (p−n-type semiconductor quenching effect), but also act as peroxidase mimetics to produce catalytic precipitate. Additionally, the steric hindrance effect from the Co3O4-Au polyhedra will decrease the PEC response of the Bi2S3. Ingeniously, the precipitates can not only deposit on the ITO electrode to decrease the photocurrent of PEC sensor, but also act as electron acceptors to scavenge the photo-generated electrons of Co3O4-Au polyhedra, leading to enhanced quenching ability of the Co3O4-Au polyhedra. When caspase-3 exists, caspase-3 can specifically recognize and cleave the biotin-DEVD-peptide, resulting in the increase of PEC response. Based on the multifunctional Co3O4-Au polyhedra, caspase-3 is detected sensitively with a linear range from 0.5 to 50 ng mL–1 and limit of detection down to 0.10 ng mL–1. The Co3O4-Au polyhedra provide a novel signal amplifier to construct PEC sensing platform and may have potential applications in bioanalysis, disease diagnostics, and 2


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clinical biomedicine.

INTRODUCTION Photoelectrochemical (PEC) sensor, as an emerging and developing analytical method, has aroused great attention in recent years, because of its low background signal and high sensitivity.1-5 Moreover, the application of electronic readout makes the PEC method be inherent simplicity, easy miniaturization, and low cost.6,7 In PEC sensing system, the signal amplification methods are the key issues and basically related to the enzymatic reactions, steric hindrance effects and p−n-type semiconductor quenching effects.8-10 For the enzymatic reactions-related PEC sensors, enzymatic catalytic precipitation strategy was received more and more attention because the insoluble precipitates formed on the electrode surface could effectively lower the detection limits.11-14 For instance, Zhao et al. developed a highly sensitive PEC immunosensor based on horseradish peroxidise (HRP) induced biocatalytic precipitation.11 However, natural enzymes usually have some defects, such as expensive cost, poor stability, and low catalytic activity easily affected by external conditions.15 To overcome these problems, various mimetic enzymatic catalysts have been exploited.16 For instance, Gong et al. proposed a self-enhanced ultrasensitive photocathodic immunoassay based on silver iodide-chitosan nanotag induced biocatalytic precipitation method.12 Zhuang et al. reported a PEC sensor via DNAzyme-mediated catalytic precipitation amplification for sensitive assay of polynucleotide kinase activity.13 On the other hand, there are quenching effects 3



between p-type and n-type semiconductors, due to the fact that the p-type semiconductor competes with n-type semiconductor to absorb the exciting light and to consume the electron donors, leading to the significant quenching effect of p-type semiconductor to n-type semiconductor.9,10 Based on this quenching effect, several PEC sensors were developed. For example, Fan et al. developed an ultrasensitive PEC immunosensor based on TiO2/CdSeTe@CdS:Mn (n-type) sensitization structure quenched by CuS (p-type) nanocrystals.9 Li et al. proposed a new PEC sensing platform for ATP assay based on PbS (p-type) quantum dots as the quencher of fullerene−Au NP@MoS2 composite.10 It is well known that cobalt oxide (Co3O4) is a typical p-type semiconductor and has a narrow band gap of about 2.07 eV.17-19 Particularly, Co3O4 exhibits intrinsic peroxidase-like activity to catalyze the oxidation of the peroxidase substrate.20,21 For example, Co3O4 with gillyflower-like nanostructure were used to catalyze the oxidation of 3,3,5,5-tetramethylbenzidine in the presence of H2O2.20 The super peroxidase-like activity of the Co3O4 nanoparticles (NPs) coupled with carbon nitride nanotubes and reduced graphene oxide has also been demonstrated.21 On the other hand, Au NPs have good electric conductivity, superior electrocatalytic properties towards the H2O2 reduction, and fascinating localized surface plasmon resonance (LSPR) properties to promote charge separation and to enhance the photoelectric conversion efficiency of semiconductors.22,23 Furthermore, catalysts with specific morphology (such as polyhedra) and high specific surface area are beneficial to their excellent catalytic activities owing to the superior mass transfer of reactants and high 4


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active sites.24 These inspire us to synthesize Co3O4-Au polyhedra with high specific surface area and to explore their application in the construction of new PEC sensors as the PEC quencher towards n-type semiconductors and enzymatic mimetics in enzymatic catalytic precipitation strategy. Here, taking zeolitic imidazolate framework (ZIF-67) as the precursor, p-type semiconducting Co3O4-Au polyhedra with high specific surface area were synthesized. Due to the important role of caspase-3 in the apoptosis-associated study,25-27 a new p-type semiconducting Co3O4-Au polyhedral-based PEC sensor has been constructed for caspase-3 assay (Scheme 1). In this PEC sensing platform, the n-type semiconductor Bi2S3-modified indium−tin oxide (ITO) slice was used as the photoelectrode due to a direct band gap of 1.3 eV and high photon-electron conversion efficiency of Bi2S3.28-30 The Co3O4-Au polyhedra were used as the multifunctional signal amplifier to amplify the PEC response signals: 1) the p-type semiconducting Co3O4-Au polyhedra can quench the photocurrents of n-type Bi2S3 because of competitive consumption of electron donors and irradiating light energy of the PEC system (p−n-type semiconductor quenching effect); 2) the Co3O4-Au polyhedra can act as peroxidase mimetics to efficiently catalyze 4-chloro-1-naphthol (4-CN) by H2O2 to produce benzo-4-chloro-hexadienone (4-CD) precipitates on the surface of the sensor, which will obviously decrease the photocurrents (mimetic enzymatic catalytic precipitation effect); 3) this 4-CD precipitates can act as electron acceptors to enhance the quenching ability of Co3O4-Au polyhedra; 4) the steric hindrance effect from the Co3O4-Au polyhedra can retard the transport of electron 5



donor and acceptor, resulting in the decrease of the photocurrents. Based on the multifunctional Co3O4-Au polyhedra, caspase-3 is detected sensitively with a wide linear response range (0.5 - 50 ng mL–1) and a low detection limit (0.10 ng mL–1). The prepared Co3O4-Au polyhedra propose a novel signal amplifier to construct PEC sensing platform and may have potential applications in bioanalysis, disease diagnostics, and clinical biomedicine.

Scheme 1. (A) The preparation procedure of Co3O4-Au-SA. (B) The mechanism of a multifunctional p-type semiconducting Co3O4-Au polyhedral-based PEC sensor.

EXPERIMENTAL SECTION Materials and Reagents. Human recombinant caspase-3 was purchased from R&D Systems (Minneapolis, USA). Caspase-3 Inhibitor (Ac-DEVD-CHO) was obtained from Beyotime Institute of Biotechnology (Haimen, China). The 6


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biotin-DEVD-peptide (biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Cys) was obtained from GL Biochem. Ltd. (Shanghai, China). Streptavidin (SA) was obtained from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The ITO slices were purchased from Zhuhai Kaivo Electronic Components Co., Ltd, China. Dithiothreitol (DTT), tris-(2-carboxyethyl)

phosphine

hydrochloride

(TCEP),

chloroauric

acid

(HAuCl4·4H2O) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (USA). Bismuth nitrate (Bi(NO3)3·5H2O), cobalt nitrate hexahydrate (Co(NO3)2·6H2O), trisodium citrate dehydrate (Na3C6H5O7·2H2O), sodium sulfide (Na2S·9H2O), 4-chloro-1-naphthol (4-CN), 2-methylimidazole, methanol (CH3OH), ascorbic acid (AA) and hydrogen peroxide (H2O2) were all bought from Sinopharm Chemical Reagent Co., Ltd. (China). Other chemicals were of analytical grade and used as received. All solutions were prepared with ultrapure water from a Milli-Q filtration system (USA). Apparatus. Scanning electron microscopy (SEM, JSM-6700F, Japan) and transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, Holland) were used to characterize the morphology and structure of the materials. The UV-vis spectra were tested using a UV-vis spectrophotometer (UV-2500, LabTech). Powder X-ray diffraction (PXRD) was recorded on an X-ray diffractometer (PXRD, D/MAX-RA,

Japan).

Nitrogen

adsorption–desorption

isotherms

and

Brunauer–Emmett–Teller (BET) surface area of the material were determined by an ASAP 2020 Micrometrics sorptometer (USA). The light source was a Xe lamp (PLS-SXE300) with a 420 nm filter. Electrochemical impedance spectroscopy (EIS) 7



and PEC measurements were measured on a CHI 660D electrochemical workstation. A three-electrode system consisted of a modified ITO electrode (5.6 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the auxiliary electrode. Preparation of Bi2S3 NPs. 0.020 mmol Bi(NO3)3·5H2O and 0.035 mmol Na2S·9H2O were dissolved in CH3OH (5 mL), respectively. Then Bi(NO3)3 solution was added into Na2S solution. The resultant mixture solution was stirred at room temperature for 2 h. Finally, Bi2S3 NPs were collected by centrifugation, and then the black precipitate was dispersed with ultrapure H2O to form Bi2S3 suspension (2 mg mL–1). Preparation of Co3O4-Au polyhedra. According to the previous work, ZIF-67-derived Co3O4 polyhedra were synthesized.31 The detail procedure was presented in the Supporting Information. For the preparation of Co3O4-Au polyhedra, 50 mL HAuCl4 solution (1 mM) was poured into a 100 mL round flask, then Co3O4 polyhedra (50 mg) were added and stirred at room temperature. Subsequently, the mixture was heated to boiling. Then, 5 mL Na3C6H5O7 solution (38.8 mM) was quickly poured under vigorous stirring for 10 min. The obtained Co3O4-Au polyhedra were washed by water for several times and re-dispersed with water to form Co3O4-Au polyhedra aqueous suspension (1 mg mL–1).

The Co3O4-Au-SA polyhedra were prepared via the conjugation between NH2 groups of SA and Au of the Co3O4-Au polyhedra.32 Typically, 100 µL of SA (1 mg

8


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mL–1) was injected into 500 µL of the Co3O4-Au polyhedra aqueous suspension (1 mg mL–1) and the mixture was slightly stirred for 12 h at 4 °C. After washed with PBS (pH 7.4, 0.1 M) to remove the unbound SA, the Co3O4-Au-SA polyhedra were obtained. Finally, the Co3O4-Au-SA polyhedra were re-dispersed in 500 µL of PBS (pH 7.4) containing 1% BSA and kept at 4 °C for future use. Construction of the PEC sensor and PEC measurement. Before modification, ITO electrode was washed with acetone, 1.0 M NaOH ethanol/water (v/v, 1:1) solution and H2O in an ultrasonic cleaner, respectively, and dried at 60 °C for 2 h. Subsequently, 25 µL of Bi2S3 suspension was coated on the cleaned ITO electrode and dried, the ITO/Bi2S3 electrode was gained. Before immobilization onto the ITO/Bi2S3 electrode surface, the biotin-DEVD-peptide was dissolved in 0.1 M acetate buffer solution (containing 10 mM TCEP, pH 5.2) and incubated for 1 h to prevent the cysteine from forming disulfide bonds. Then, 20 µL of biotin-DEVD-peptide solution (0.1 mM) was dripped onto the ITO/Bi2S3 electrode surface for 12 h at 4 °C to immobilize the peptide on the electrode via the chemical interaction of -SH from Cys with the exposed, unsaturated Bi3+ at the Bi2S3 surface.33 After washed with acetate buffer (pH 5.2), the obtained ITO/Bi2S3/biotin-DEVD-peptide electrode was infiltrated into 1 wt% BSA for 1 h to reduce the non-specific adsorption. After rinsing with PBS (pH 7.4), the obtained electrode was further incubated with assay buffer (20 µL, PH 7.4, 25 mM HEPES, 0.1% CHAPS and 10 mM DTT) containing different concentrations of caspase-3 for 30 min at 37 °C. Next, the electrode was incubated with 20 µL of Co3O4-Au-SA polyhedra suspension for 2 h at 37 °C to introduce 9



Co3O4-Au-SA polyhedra to the sensor via the specific interaction between biotin and SA. To carry out the mimetic enzymatic catalytic precipitation (MECP) reaction, the obtained electrode (ITO/Bi2S3/biotin-DEVD-peptide/Co3O4-Au-SA) was incubated with 10 mM 4-CN solution containing 1 mM H2O2 for 20 min at room temperature. After that, the PEC measurements were carried out in PBS (pH 7.4, 0.1 M) containing 0.1 M AA at 0 V.

RESULTS AND DISCUSSION Characterization of Co3O4 and Co3O4-Au polyhedra. The SEM images of the ZIF-67, Co3O4 and Co3O4-Au polyhedra are displayed in Figure 1. It is noted that the ZIF-67 polyhedra (Figure 1A) have rhombic dodecahedral shape with the average size of about 550 nm.31 After calcination of the ZIF-67 polyhedra in air, the Co3O4 polyhedra (Figure 1B) retain the similar morphology, although the average size of Co3O4 polyhedra reduces to half of that of ZIF-67 polyhedra. From Figure 1C, it can be obtained that Au NPs are dispersed on the surface of Co3O4 polyhedra. The successful preparation of Co3O4 polyhedra is further confirmed by the powder X-ray diffraction (PXRD) results. From Figure 1D, all the diffraction peaks of ZIF-67 polyhedra (curve a) are similar to those reported previously.34 After the calcination process, the diffraction peaks of ZIF-67 polyhedra entirely disappear (curve b), and five new peaks, corresponding to (220), (311), (400), (511), and (440) planes, can be observed at 31.3°, 36.9°, 44.8°, 59.3°, and 65.5° (curve b), respectively, which indicates the successful synthesis of Co3O4 (JCPDS no. 42-1467).35 Additionally, the 10


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nitrogen adsorption-desorption isotherms of the Co3O4 polyhedra are obtained (Figure 1E). From Figure 1E, the Co3O4 polyhedra have porous structure and the Brunauer−Emmett−Teller (BET) surface area of Co3O4 polyhedra is measured to be 100.9 m2 g−1. Based on the high specific surface area, polyhedral morphology and porous structure, Co3O4 polyhedra can supply sufficient catalytic active sites and facilitate the transfer of molecules and ion diffusion.36,37 Moreover, the UV−vis absorption spectra of Co3O4-Au and Co3O4 polyhedra are displayed in Figure 1F. Compared with the Co3O4 polyhedra (curve a), the Co3O4-Au polyhedra (curve b) show a typical surface plasma resonance absorption of Au NPs at 520 nm, further indicating that Au NPs are successfully modified on the Co3O4 polyhedra.38

11



Figure 1. SEM images of (A) ZIF-67 polyhedra, (B) Co3O4 polyhedra, (C) Co3O4-Au polyhedra. (D) PXRD patterns of (a) ZIF-67 polyhedra and (b) Co3O4 polyhedra. (E) Nitrogen adsorption-desorption isotherms of Co3O4 polyhedra. (F) UV-vis absorption spectra of (a) Co3O4 polyhedra and (b) Co3O4-Au polyhedra. Inset of D is the related pore size distribution. 12


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EIS and PEC Characterization of the developed PEC sensor. Due to the narrow band gap and high photon-electron conversion efficiency,28-30 Bi2S3 NPs were used as photoactive materials and Bi2S3 NPs-modified ITO slices were used as photoelectrode. The SEM, TEM and high-resolution TEM (HRTEM) images of the as-synthesized Bi2S3 NPs are shown in Figure 2. It is noted that Bi2S3 NPs have a particle size of about 5 nm and are aggregated. From Figure 2B, the lattice fringes with spacing of 0.316 and 0.331 nm can be evidently observed, which are indexed to the (211) and (021) planes of Bi2S3 (JCPDS no. 89-8965), respectively. From the insert of Figure 2A, the UV-vis absorption spectrum of Bi2S3 NPs exhibits a broad absorption range and effective visible absorption intensity, indicating that Bi2S3 NPs should be suitable n-type semiconducting materials to construct PEC sensing platform.

Figure 2. (A) SEM and (B) HRTEM images of Bi2S3 NPs. The inset plots are the UV-vis absorption spectrum (A) and TEM image (B) of Bi2S3 NPs, respectively.

EIS is an effective approach for investigating the fabrication of the electrode. The semicircle diameter from EIS measurements reveals the interfacial charge-transfer 13



resistance (Rct) of the electrode.39 Figure 3A shows that the ITO/Bi2S3 electrode has a small Rct (115 Ω, curve a). Nevertheless, after the stepwise assembling of biotin-DEVD-peptide and BSA in order, the Rct values are increased to 194 Ω (curve b) and 229 Ω (curve c), due to the non-conductive properties of peptide and protein molecules. However, when the electrode (ITO/Bi2S3/biotin-DEVD-peptide/BSA) is further reacted with caspase-3, the peptide is specifically cleaved by caspase-3, resulting in a significant decreasing Rct value (191 Ω, curve d). After incubation with streptavidin-labeled Co3O4-Au (Co3O4-Au-SA) solution, the Co3O4-Au-SA polyhedra are introduced to the sensing platform based on the interaction between biotin on the uncleavaged DEVD-peptide and SA on the Co3O4-Au polyhedra. This results in an increased Rct value (233 Ω, curve e), due to the steric hindrance effect from the semiconducting Co3O4-Au polyhedra. Additionally, the Rct value of the resulting electrode increases dramatically after incubation with 4-CN and H2O2 mixture (338 Ω, curve f). This mainly contributes to the fact that the insoluble 4-CD deposits on the electrode to obstruct the electron transfer of [Fe(CN)6]3−/4− at the electrode surface. Here, Co3O4 acts as peroxidase mimics to efficiently catalyze the formation of 4-CD precipitates from 4-CN in the presence of H2O2. As reported previously,40 Au NPs could also catalyze the formation of 4-CD from 4-CN. The possible catalytic mechanism is as follows:41-44 a hydroxyl radical (·OH) is generated in the H2O2 solution in the presence of Co3O4-Au polyhedra and the resultant ·OH reacts with 4-CN to form the insoluble 4-CD which deposits on the electrode. The EIS results indicate that the designed caspase-3 sensor is satisfactorily fabricated according to 14


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Scheme 1B.

Figure 3. (A) EIS in 0.1 M KCl solution containing 5 mM (1:1) [Fe(CN)6]3−/4−. Amplitude, 5 mV; frequency range, 0.1 - 100 kHz. (B) photocurrent responses in PBS (0.1 M, pH 7.4, 0.1 M AA) at 0 V. (a) ITO/Bi2S3, (b) ITO/Bi2S3/biotin-DEVD-peptide, (c) ITO/Bi2S3/biotin-DEVD-peptide/BSA, (d) ITO/Bi2S3/biotin-DEVD-peptide/BSA after cleavage by 100 ng mL–1 caspase-3, (e) ITO/Bi2S3/biotin-DEVD-peptide/BSA (cleaved

by

caspase-3)

after

incubation

with

Co3O4-Au-SA,

(f)

ITO/Bi2S3/biotin-DEVD-peptide/BSA/Co3O4-Au-SA (cleaved by caspase-3) after incubation with 10 mM 4-CN and 1 mM H2O2 for 20 min.

To further investigate the stepwise fabrication process, the electrode is also characterized by the PEC method. From Figure 3B, the ITO/Bi2S3 electrode shows a large photocurrent (curve a), due to the excellent photoelectric activity of Bi2S3. Subsequent incubation with the biotin-DEVD-peptide and BSA, the photocurrents obviously decrease (curves b−c). However, the photocurrent increases obviously after cleavage of the biotin-DEVD-peptide by caspase-3 (curve d). The results are in conformity to these of EIS (Figure 3A). In addition, the capture of the Co3O4-Au-SA 15



leads to the decrease of the photocurrent (curve e). The reasons may be as follows: the Co3O4-Au polyhedra not only retard the transport of electron donors (AA) to the electrode surface (steric hindrance effect), but also as quencher to effectively weaken the photocurrents of the electrode because of the competitive consumption of electron donors (AA) and light energy (p−n-type semiconductor quenching effect).9,10 At last, the photocurrent of the developed sensor significantly decreases after incubated with the 4-CN and H2O2 mixture, suggesting that the Co3O4-Au polyhedra (peroxidase mimetics) have good catalytic activity for accelerating the formation of 4-CD precipitates from 4-CN in the presence of H2O2. And the precipitates not only serves as electron acceptors to improve quenching ability of the Co3O4-Au polyhedra (as shown in Figure S1, see the Supporting Information), but also as insoluble precipitates to hinder AA (electron donor) to reach the electrode surface.12 These results demonstrate that the Co3O4-Au polyhedra-based PEC sensing platform has been manufactured successfully according to Scheme 1 and can be used in caspase-3 activity assay. Optimization of experimental conditions. In order to achieve the good analytical performance and optimum state of the proposed sensor for caspase-3 assay, several experiment parameters, including the biotin-DEVD-peptide concentration, the reaction time between caspase-3 and biotin-DEVD-peptide, and the catalytic deposition time of 4-CN were optimized. The effect of the concentration of biotin-DEVD-peptide on the photocurrent of the ITO/Bi2S3/biotin-DEVD-peptide electrode was investigated. From Figure 4A, the 16


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photocurrent decreases with the increasing biotin-DEVD-peptide concentration owing to the non-conductive property of peptide. A plateau is reached at 0.1 mM, implying the saturated immobilization of the biotin-DEVD-peptide. So, 0.1 mM is taken as the optimal biotin-DEVD-peptide concentration. The reaction time between caspase-3 and biotin-DEVD-peptide is a key factor and should

be

optimized.

With

the

increment

of

reaction

time,

more

biotin-DEVD-peptides are cleaved and less Co3O4-Au polyhedra are attached to the electrode, leading to the increased photocurrents. As shown in Figure 4B, a plateau reaches at 30 min. Therefore, 30 min is selected as the optimal time for the reaction between caspase-3 and biotin-DEVD-peptide. The catalytic deposition time of 4-CN is also an important parameter and optimized. From Figure 4C, the photocurrent decreases with the increasing catalytic deposition time because of the fact that the longer catalytic deposition time accumulates more 4-CD precipitates on the PEC electrode surface. It is noted that a plateau is reached at 20 min. Thus, 20 min is chosen as the catalytic deposition time of 4-CN.

Figure 4. Effects of (A) the biotin-DEVD-peptide concentration, (B) the reaction time between caspase-3 and biotin-DEVD-peptide (biotin-DEVD-peptide, 0.1 mM; caspase-3, 100 ng mL–1) and (C) the catalytic deposition time of 4-CN 17



(biotin-DEVD-peptide, 0.1 mM; caspase-3, 100 ng mL–1; caspase-3 reaction time, 30 min) on the photocurrents of the modified electrode.

Caspase-3 activity assay. Under optimal conditions, the PEC sensor is developed for caspase-3 assay. From Figure 5A, the photocurrents increase with the increasing concentration of caspase-3 (0 - 200 ng mL–1). The calibration curve (∆I = I – I0, where I0 and I are photocurrents of the sensor incubated without and with caspase-3, respectively) are shown in Figure 5B. The linear response range is from 0.5 to 50 ng mL–1 with the linear regression equation of ∆I (µA) = 0.12894 + 0.11272 Ccaspase-3 (ng mL–1) (R2 = 0.995). The detection limit is estimated to be 0.10 ng mL–1 (3σ), which is lower than that recently reported (Table S1, see the Supporting Information).

Figure 5. (A) Photocurrent responses of the different concentration of caspase-3. The concentrations are 0, 0.5, 1, 5, 10, 20, 30 50, 100, and 200 ng mL–1 (from a to j). (B) Dependence of ∆I on caspase-3 concentration.

Selectivity, reproducibility and stability. To investigate the selectivity of the 18


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developed PEC caspase-3 assay, several possible interfering proteins, including glucose oxidase (GOX), BSA, lysozyme, alkaline phosphatase (ALP) and pepsin are selected. From Figure 6A, the changes of photocurrent response caused by interfering proteins are very small. However, the photocurrent of the sensor toward caspase-3 (50 ng mL–1) can be obviously observed due to the fact that caspase-3 can specifically recognize and cleave biotin-DVED-peptide. In order to make sure the selectivity of the PEC sensor, caspase-3 inhibitor (Ac-DEVD-CHO) is used to inhibit caspase-3 activity. Caspase-3 is pre-incubated with 100 µM Ac-DEVD-CHO for 30 min before reaction with biotin-DEVD-peptide. As shown in Figure 6B, the photocurrent shown in column b is almost the same with that observed in column a, indicating a good inhibiting effect of Ac-DEVD-CHO on the caspase-3 activity. This also implies that the developed PEC sensor has high selectivity toward the caspase-3 activity assay and can also be used to quickly screen the related inhibitors of caspase-3. The reproducibility of the sensor is also evaluated. Five sensors prepared separately in the same conditions were used to detect caspase-3 (50 ng mL–1), and the relative standard deviation is 4.3%. Additionally, the stability of the sensor is also evaluated. The PEC response of the sensor can retain about 92.7% of its initial photocurrent response toward caspase-3 after 18 days of storage in the refrigerator at 4 °C. These demonstrate that the developed PEC sensor has satisfactory reproducibility and acceptable stability.

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Figure 6. (A) Selectivity of the PEC caspase-3 sensor. The caspase-3 concentration, 50 ng mL–1; the concentration of other proteins, such as GOX, BSA, lysozyme, ALP and pepsin, 5000 ng mL–1. (B) PEC responses for caspase-3 inhibition by Ac-DEVD-CHO. (a) in the absence of caspase-3, (b) in the presence of 50 ng mL–1 caspase-3 with 100 µM Ac-DEVD-CHO, and (c) in the presence of 50 ng mL–1 caspase-3.

Recovery test. In order to investigate the analytical applicability in complex biological system, the proposed PEC sensor was used to analyse caspase-3 in cell extracts. Different concentrations of caspase-3 (1, 5 and 50 ng mL–1) were added into the 100-fold diluted Human breast adenocarcinoma (MDA-MB-231) cell extracts (friendly provided by Nie’s group in Hunan University, diluted by 25 mM HEPES, 0.1% CHAPS and 10 mM DTT (pH 7.4)). As shown in Table S2 (see the Supporting Information), the average recoveries in cell extracts are 97, 103 and 99%, respectively, demonstrating that the proposed PEC sensor has promising applications in caspase-3 assay in real samples.

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CONCLUSIONS In summary, p-type semiconducting Co3O4-Au polyhedra were synthesized and used as the signal amplifier to construct a sensitive PEC sensor for caspase-3 activity assay. The Co3O4-Au polyhedra could not only serve as quencher to dramatically weaken the PEC response of the Bi2S3 photoelectrode because of competitive consumption of AA and light energy, but also as peroxidase mimetics to efficiently produce catalytic precipitate. Ingeniously, the produced precipitates not only cover on the sensor surface to block the PEC process, but also act as electron acceptors to enhance quenching ability of Co3O4-Au polyhedra. Additionally, the steric hindrance effect from the Co3O4-Au polyhedra further decreases the PEC response of the sensor. The developed PEC sensor exhibits a wide linear response range (0.5-50 ng mL–1) and a low detection limit (0.10 ng mL–1) for caspase-3 assay. What’s more, the strategy possesses good selectivity, satisfactory reproducibility and acceptable stability. The developed multifunctional p-type semiconducting Co3O4-Au polyhedra provide a new signal amplifier for PEC bioanalytical applications and stimulate the exploitation of other multifunctional semiconductors as photoactive enzyme mimetics.

AUTHOR INFORMATION Corresponding Author *Tel./Fax: +86 731 88821961. E-mail: [email protected] Notes The authors declare no competing financial interest. 21



ACKNOWLEDGMENTS This work was financially supported by NSFC (21727810, 21475035, 21235002), and the Foundation for Innovative Research Groups of NSFC (21521063). Supporting Information Preparation of Co3O4 polyhedra, PEC characterization of Co3O4-Au polyhedra, comparison of different methods for caspase-3 assay, recovery test. This material is available free of charge via the Internet at http://pubs.acs.org.

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Co3O4âAu Polyhedra: A Multifunctional Signal Amplifier for Sensitive

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