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A Paper-Based Device for Colorimetric and Photoelectrochemical Quantification of the Flux of H2O2 Releasing from MCF-7 Cancer Cells Li Li, Yan Zhang, Lina Zhang, Shenguang Ge, Haiyun Liu, Na Ren, Mei Yan, and Jinghua Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00693 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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Analytical Chemistry

A Paper-Based Device for Colorimetric and Photoelectrochemical Quantification of the Flux of H2O2 Releasing from MCF-7 Cancer Cells Li Li,a Yan Zhang,a Lina Zhang,b Shenguang Ge,a,b Haiyun Liu,a Na Ren,c Mei Yan,a and Jinghua Yu*a

a

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022,

P.R. China. b

Shandong Provincial Key Laboratory of Preparation and Measurement of Building

Materials, University of Jinan, Jinan 250022, P.R. China. c

School of Biological Science and Technology, University of Jinan, Jinan 250022, P.R.

China.

Abstract In this work, a novel dual photoelectrochemical (PEC)/colorimetric cyto-analysis format was first introduced into a microfluidic paper-based analytical device (µ-PAD) for synchronous sensitive and visual detection of H2O2 released from tumor cells based on an in situ hydroxyl radicals (·OH) cleaving DNA approach. The resulted µ-PAD offered an excellent platform for high performance biosensing applications, which was constructed by a layer-by-layer modification of concanavalin A, graphene quantum dots (GQDs) labeled flower-like Au@Pd alloy NPs probe and tumor cells on the surface of the vertically aligned bamboo like ZnO, which grows on a pyknotic Pt nanoparticles (NPs) modified paper working electrode (ZnO/Pt-PWE). It was the

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effective matching of energy levels between GQDs and ZnO levels that lead to the enhancement of the photocurrent response compared with the bare ZnO/Pt-PWE. After releasing H2O2, the DNA strand was cleaved by ·OH generated under the synergistic catalysis of GQDs and Au@Pd alloy NPs and thus, reduced the photocurrent, resulting in a high sensitivity to H2O2 in aqueous solutions with a detection limit of 0.05 nmol observed, much lower than that in the previously reported method. The disengaged probe can result in catalytic chromogenic reaction of substrates, resulting in real-time imaging of H2O2 biological processes. Therefore, this work provided a truly low-cost, simple and disposable µ-PAD for precise and visual detection of cellular H2O2, which had potential utility to cellular biology and pathophysiology. Keywords: Dual photoelectrochemical/colorimetric cyto-analysis; µ-PAD; Visual detection; Cellular H2O2

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1. Introduction Hydrogen peroxide (H2O2), formed by disproportionation of unstable reactive oxygen species superoxide ions (O2-), is one of the major contributors to the oxidative stress damage because of its long lifetime to diffuse to other cellular compartments.1-2 However, to date, it has also been acknowledged as a messenger molecule related to cell proliferation, migration, and differentiation in a concentration dependent manner.3-4 For this reason, the development of efficient methods for detection of intracellular H2O2 is critical importance in elaborating its regulation of signal transduction pathways and searching for new therapeutic strategies for diseases. In recent years, a variety of fluorescence methods,5 chemiluminescence,6 and electron spin resonance7 etc., have been developed to assay cellular H2O2. Although these methods give relatively low detection limits, they are not suitable for point-of-care (POC) diagnosis, since these analytical systems were dependent on specific infrastructures which were normally pricy, large and complicated. Mounting colorimetries based on the specific high activity of natural enzymes towards catalyzing a chromogenic substrate to form the colored product have also been mostly reported.8-9 However, the inherent drawbacks of the natural enzyme are that the enzymes would not maintain their activity under the detection conditions, which limits its application in biosensors.10-12 As a result, there is a strong need to develop a method, which could not only solve the problems of the low long-term operational stability, but also have the abilities for realizing simple, visual and reliable detection of cellular H2O2.

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Paper, as one of the four great inventions of ancient China, was wildly used as a platform for the development of simple and cost-effective molecular diagnostic assays. To date, the paper-based colorimetric detection device using variety of enzyme-mimicking toward the chemical or electrochemical reduction of H2O2 have primarily used for the qualitative analysis of polytype analytes13 due to their simplicity and low-cost and often visual. Besides, as a newly developed promising analytical

method

for

the

detection

of

biomolecules,

paper-based

photoelectrochemicals (PEC) sensor devices possesses the advantages of high selectivity and sensitivity because of the separation of the excitation source and the detection signal.14-15 In this work, we first developed a microfluidic paper-based dual PEC/colorimetric cellular analytical devices (µ-PEC/C), which combine the advantages of the usefulness of PEC detection for quantitative analysis with colorimetric detection for screening. The feasibility of this device was confirmed experimentally with the determination of H2O2 released from MCF-7 cells. Graphene quantum dots (GQDs), as recently emerging carbon-based materials, are graphene sheets smaller than 100 nm.16 GQDs have attracted more and more attention due to its unique properties, such as lower toxicity, good biocompatibility, excellent solubility, higher peroxidase-like activity. Besides, GQDs possess excellent optical and electronic properties, and in particular possess a bandgap less than 2.0 eV because of quantum confinement and edge effects.17-19 Given the features mentioned above, GQDs shows promising nanomaterials, which can be exploited to improve colorimetric or optical sensing performances.20-21 To achieving highly sensitive

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cyto-assays on the µ-PEC/C method using GQDs as a green sensitizer and catalyst, signal amplification strategies was necessary. In this work, flower-like Au@Pd (FL-Au@Pd) alloy NPs with large specific area, superior conductivity and high catalytic efficiency towards the decomposition of H2O222 was first synthesized and used as carriers for GQDs by noncovalent interaction via a single strand DNA mediate function. On the other hand, a dense Pt NPs layer was grown on the surfaces of paper fiber in the paper working electrode (Pt-PWE) and used as sensor platform for subsequently vertically aligned bamboo like ZnO growing (BL-ZnO/Pt-PWE). This work presents a novel signaling amplification strategy for dual PEC/C analytical application and to the best of our knowledge has never been reported. Herein, we report a novel and versatile µ-PEC/C cyto-sensing platform based on the exquisite integration of an intriguing PWE modified with large rough surface area BL-ZnO and good electric conductors Pt NPs with the versatile function of the GQDs and Au@Pd alloy NPs. Specifically, the vertically aligned BL-ZnO, with low losses in charge recombination and fast vectorial charge transport perpendicular to the charge-collecting substrates,23-26 accepted electrons from the illuminated GQDs and transfered immediately to the Pt-PWE, which resulted in improved charge separation and an obviously increased photocurrent of BL-ZnO. However, when the presence of H2O2, FL-Au@Pd alloy NPs and its supported numerous GQDs (refer to as probe agents) catalyze the decomposition of H2O2, generating ·OH. As a result, a good deal of DNA molecules were cleaved by ·OH, thus resulting many probe agents far away from the surface of the PWE, leading to the decrease of the photocurrent signal. The

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dissociative probe agents were transfered outward to the colorimetric detection zones as the electrolyte solution flows and catalyzed a chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) to form the colored product. This work opens a different perspective for transducer design in PEC detection and provides a novel format in sensing H2O2 changes in biological systems. 2. Materials and methods 2.1. Reagents and materials All chemicals and solvents related to cell culture and cell testing were aseptic. Chloroplatinic acid (H2PtCl6), Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and ammonium acetate (CH3COONH4) were obtained from Shanghai Chemical Reagent Company (Shanghai, China). 4-Aminothiophenol (PATP) was obtained from Acros Organics Chemical Co. Ascorbic acid (AA, ≥99.0%), bovine serum albumin (BSA), and concanavalin A (Con-A), phorbol myristate acetate (PMA, ~99%, Sigma), adenosine 5’-diphosphate (ADP, ≥95%), N-formylmethionyl-leucylphenylalanine (fMLP, ≥97%) were obtained from Sigma-Aldrich Chemical Co. (USA). Solutions of H2O2 were freshly diluted from the 30% solution, and their concentrations were determined using a standard KMnO4 solution. Whatman chromatography paper #1 (58.0 cm × 68.0 cm) (pure cellulose paper) was obtained from GE Healthcare Worldwide (Pudong Shanghai, China) and used with further adjustment of size (A4 size). All chemicals and solvents were used as received with the analytical grade or above. Ultrapure water obtained from a Millipore water purification system (>18.2 MΩ.cm, Milli-Q, Millipore) was used in all assays and solutions.

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2.2. Preparation of GQDs The GQDs was prepared from Vulcan XC-72 carbon black by refluxing with concentrated nitric acid.27 Briefly, 0.25 g of XC-72 carbon black was added into 50 mL of 6 mol·L − 1 HNO3. After sonicated for 1 h, the suspension was refluxed for 24 h at 130 °C. After cooling to room temperature, the suspension was centrifuged for 30 min at 8000 rpm to obtain a yellow supernatant. The supernatant was heated at 100 °C to remove water and nitric acid, and the GQDs were obtained as a reddish-brown solid. Subsequently, the solid was further dialyzed in a dialysis bag for 3 days. After dialysis, the solution was centrifuged for 30 min. Finally, the GQDs were kept at 4 °C for further use. 2.3. Preparation of GQDs/FL-Au@Pd conjugated DNA strands The FL-Au@Pd alloy NPs were prepared via a facile wet-chemical reduction route by using H2PdCl4 and HAuCl4 as metal sources and AA as reductant in the presence of octadecyl

trimethyl

ammonium

chloride

(OTAC)

and

copper-(II)

acetate

(Cu(CH3COO)2) according to the reported literature28 with slight modifications and the procedure was described below. An H2PdCl4 aqueous solution (2.0 mL, 20.0 mmol·L-1), a OTAC glycol solution (0.05 mL, 0.10 mol·L-1) and a Cu(CH3COO)2 aqueous solution (0.05 mL, 1.0 mmol·L-1) were added into an aqueous HAuCl4 solution (3.0 mL, 1.0 mmol·L-1) in order. After homogeneous mixing, a freshly prepared AA aqueous solution was quickly added with a gentle shaking to this solution and left undisturbed for 12 hours at 15 °C. Thiol-DNA modified FL-Au@Pd alloy NPs was obtained according to the

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previous report.29-30 For the immobilization of GQDs, DNA modified FL-Au@Pd alloy NPs (Apt/FL-Au@Pd) was redispersed in 50 mM Tris-acetate buffer (containing 300 mM NaCl, pH 7.0) and GQDs solution (10 µg·mL-1) was added. The solution was incubated at room temperature for 2 h to prepare GQDs/Apt/FL-Au@Pd via the strong π-π stacking between the DNA strand and GQDs. Excessive reagents were removed by centrifuging at 8000 rpm for 15 min. 2.4. Fabrication of the PEC detection areas The PEC detection areas were prepared according to our previous work with slight modification31 and the detailed fabrication procedure is represented in the supporting information. As shown in Scheme 1, this µ-PEC/C, comprised of one channel tab, one detection tab and one reference tab, was fabricated through wax-printing (Scheme 1). The unprinted hydrophilic area on the detection tab, containing one PEC working zone for screen-printed carbon working electrode, one pretreatment zone and one colorimetric detection zone to achieve chromogenic reactions. The other pretreatment zone and colorimetric detection zone was used as an evidence that the chromogenic reactions went through a radical chain mechanism (Scheme 1B). After folding at the predefined fold line (1 mm in width), the three screen-printed electrodes would be connected once the paper cell was filled with solution (Scheme 1C). The corresponding detection mechanism was shown in Scheme 2. The Pt-PWE with enhanced conductivity and enlarged surface area was fabricated by using H2PtCl6 as metal sources and NaBH4 as reductant. Briefly, freshly prepared growth aqueous solution (15 µL) containing H2PtCl6 (200 mM) and NaBH4 (20 mM) was added into the surface of the bare PWE, keeping it at room temperature for approximately 10 min. Following that, the resulting Pt-PWE was washed with

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water thoroughly and dried at room temperature for 20 min before use.

Scheme 1 The schematic representation, size, and shape of the 3D paper-based device. Paper sheets were firstly patterned in bulk using a wax printer. Then electrodes were screen-printed on A4 paper sheet in bulk. After laser cut and modification, the device was used to detect H2O2 released from tumor cells. 9



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The BL-ZnO NPs was synthesized via a simple electrodepositing method, consisting of a seeding step followed by the growth step. Firstly, prepared ZnO seeds solution32 was dropped onto the Pt-PWE and spin-coated at 1000 rpm for 60 s. After that, the Pt-PWE, covered with a dense ZnO seed layer, was dried at 100 °C for 10 min. The coating and heating cycle was repeated six times. Subsequently, BL-ZnO was formed on the Pt-PWE by immersion into an aqueous solution consisting of 50 mM Zn(NO3)2·6H2O, 50 mM CH3COONH4, 70 mM ethylenediamine at 70 °C electrodeposition for 12 h and then thoroughly rinsed with Milli-Q water and anhydrous alcohol, the BL-ZnO arrays were obtained. To obtain Con-A modified ZnO/Pt-PWE, 10 µL of Con-A aqueous solution (5 mM) was added to the surface of the ZnO/Pt-PWE and incubated for 40 min at room temperature, following by washing with the washing buffer. For immobilization of the GQDs/Apt/FL-Au@Pd on the surface of ZnO/Pt-PWE, 10 µL PATP solution (0.1 mmol·L-1) was dropped onto the ZnO/Pt-PWE and incubated for 4 h, after washing with ethanol, 10 µL mixture solution containing GQDs/Apt/FL-Au@Pd and 2.5% GA was added and incubated for 50 min at room temperature. After washing with the washing buffer, each paper working zone was blocked by adding 4.0 µL of 2 mM 6-mercapto-1-hexanol (MCH) to block possible remaining cell-binding sites, and allowing the paper working zone to dry for 20 min at room temperature (Scheme 2). In this process, PATP molecules were connected to ZnO due to the formation of Zn-S bond between the thiol group of the PATP molecules and ZnO33 and the remaining amino groups of PATP molecules could bind

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to the Apt. For cell capture, 10 µL of homogeneous cell suspension at a certain concentration was dropped onto the surface of the treated Pt-PWE and incubated at room temperature for 1 h to capture the cells through the specific recognition of

Scheme 2 Schematic representation of the fabrication procedures for the PEC detection zone and PEC detection mechanism (A) and colorimetric detection mechanism (B) of H2O2 released from MCF-7 cells.

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mannose on the captured cell surfaces by Con-A (Scheme 2A). Then the electrodes were taken out and rinsed with incubation buffer to remove the noncaptured cells. Finally, the resulting PEC was stored at 4 °C prior to use. 2.5. Construction of the colorimetric device The fabrication procedure for the colorimetric detection zones is as follows: in brief, 20 µL of 20 mM TMB were added to the colorimetric detection zones. One of the pretreatment zone was added 10 µL of 5 mM H2O2 and incubated for 20 min at 25 °C and the other one was added 10 µL of 5 mM H2O2 and thiourea aqueous solution (1.5 M), which has capabilities to scavenge ·OH; therefore, they are able to inhibit the color reaction.34 2.6. Assay protocol First of all, 10.0 µL PBS solution (pH 7.4) containing PMA (200 ng·mL-1) was dropped into the paper PEC cell and then incubated at 37 °C for 2 min to cleavage of DNA strand. Subsequently, the channel tab was placed over the detection tab. Then, 30 µL 0.2 mM HAc-NaAc buffer (pH 4.6) was added into the paper PEC cell and incubated for 30 min at 30 °C (Scheme 1C, number 2). After the chromogenic reaction was finished, unfolded the channel tab. Subsequently, the PEC cell was washed with water thoroughly and then folded the reference tab down below the detection tab successively. Then, the modified 3D µ-PEC/C cyto-device was fixed and connected to the electrochemical work station (Scheme 1C, number 3). Subsequently, PBS solution (pH 7.4, 40 µL) containing AA (0.1 M·L-1) was dropped into the paper PEC cell, followed by the respective PEC measurements.

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3. Results and discussion 3.1. Structure characterization Figure 1A and Figure 1B show the SEM images of the as-prepared Pt-PWE and ZnO/Pt-PWE, respectively. As displayed in Figure 1A, a continuous and thin Pt layer was uniformly distributed on the surface of paper fiber, which was beneficial for enhancing the surface area of the PWE to grow numerous PEC active materials and accelerating the photogenerated electrons transfer. From Figure 1B we can see that the BL-ZnO grow densely on the surfaces of the fibers in their radial direction. To clearly observe the morphology of the growing BL-ZnO, high magnification SEM was used

Figure 1. SEM images of Pt-PWE (A) and ZnO/Pt-PWE (B); Enlarged ZnO NPs on the surfaces of Pt-PWE (C) under different magnifications; (D) XRD patterns of the (a) bare PWE, (b) Pt-PWE, (c) ZnO/Pt-PWE.

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and the obtained image was shown in Figure 1C. As shown in Figure 1C, the prepared ZnO have a BL structure, which had never reported on the other groups. This unique structure could greatly increase the specific surface area of the ZnO, which is beneficial for PEC signal enhancement. Besides, the crystal structures of ZnO/Pt-PWE, Pt-PWE and bare PWE were characterized by XRD (Figure 1D). In contrast to the XRD pattern of bare PWE (curve a), Pt-PWE shows two reflections at 40, 46.8º (curve b), which matched well with the Pt standard patterns (JCPD NO. 00-004-0802), suggesting that the successful preparation of Pt-PWE. The XRD pattern of ZnO/Pt-PWE (curve c) contained the diffraction peaks of PWE, Pt and ZnO NPs, indicating that the ZnO/Pt-PWE was successfully synthesized.

Figure 2. SEM (A) and TEM (B) images of FL-Au@Pd NPs, Inset: enlarged FL-Au@Pd NPs; (C) TEM image of GQDs/Apt/FL-Au@Pd. (D) XRD patterns of Au, Pd and FL-Au@Pd NPs. 14


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The morphology of the as-prepared FL-Au@Pd NPs was presented in Figure 2A. It was clearly found that a FL structure of Au@Pd NPs was obtained from the SEM image. TEM shows that the collected Au@Pd NPs are monodisperse and have a ragged surface with diameter of ca. 500-700 nm (Figure 2B). The size distribution of the FL-Au@Pd NPs was shown in Figure S5. After GQDs was linked to the surface of the FL-Au@Pd NPs, we can observe satellites assembly formed with multiple GQDs surrounding Au@Pd NPs in TEM images, as shown in Figure 2C. The crystal structure of FL-Au@Pd NPs was characterized by XRD. Figure 2D showed that four reflections appeared at 39.4, 45.8, 66.2 and 78.8º, which were coincidently located between Au (reference code: 00-004-0784) and Pd (reference code: 00-005-0681), indicating the successful fabrication of Au@Pd alloy NPs. Figure 3A showed the TEM image of GQDs. As shown in Figure 3A, the diameters of GQDs were mainly distributed in the range of 4-7 nm with average diameter of 6.5 nm. Furthermore, to further explore the optical properties of the GQDs, PL and UV-vis absorption spectra were studied (Figure 3B). The UV-vis absorption spectrum exhibits that the GQDs have a broad absorption below 600 nm with a peak at 228 nm. Their emission wavelengths are excitation-dependent, exhibiting a red shift with increasing excitation wavelength, which might be caused by the different emissive sites of GQDs.35 Fourier transform infrared (FTIR) of the GQDs was also investigated (Figure 3C). In the FTIR spectra, the GQDs showed C-OH group (alkoxy) stretching peak at 1113 cm-1 and C–O (carboxy) deformation peak at 1389 cm-1 and O-H group (carboxy) stretching peak at 3458 cm-1, showing

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that the obtained GQDs contain –COOH and –OH groups. The Raman spectrum of GQDs (Figure 3D) indicates that the GQDs have two prominent Raman features, a D band at around 1355 cm− 1 and a G band at around 1580 cm− 1. Furthermore, the XPS

Figure 3. (A) TEM and (B) UV-vis absorption and PL emission spectra of GQDs; Insets in A: photographs of the GQDs aqueous solution taken under visible light and 365 nm UV light; (C) FTIR spectra of the prepared GQDs; (D) Raman spectra of the obtained GQDs; (E) XPS analysis surveys of GQDs; (F) The XPS C1s analysis of GQDs.

of the GQDs showed three types of carbons: graphitic carbon (C=C and C-C), oxygenated carbon (C=O and C-O-C) and nitrogenated carbon (C-N). The nitrogen of GQDs originated from HNO3 oxidation during the preparation (Figure 3E and 3F), which were consistence with the previous works.[36] 3.2. The DNA cleavage mechanism analysis To test the DNA cleavage mechanism, PEC measurements of different process were recorded (Figure 4A). As expected, after BL-ZnO NPs were grown onto the Pt-PWE surface (curve a), an increase signal was observed (curve b). The photocurrent 16


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intensity further increased after Con-A, GQDs/Apt/FL-Au@Pd and cells were assembled onto BL-ZnO/Pt-PWE (curve c). Clearly, the great improvement revealed the successful GQDs/Apt/FL-Au@Pd loading. In this process, GQDs was irradiated to produce electron–hole pairs by exciting the electrons from the valence band to the conduction band. Since the energy levels between the conduction bands of ZnO and GQDs matched well, the excited electron coming from the GQDs transfer to the conduction band of ZnO readily and subsequently reaches the Pt-PWE surface, leading to the photocurrent increase. However, when 200 ng·mL-1 PMA was added into the supporting electrolyte, a modest decrease in the PEC response was observed in curve d. In this process, DNA-bridge was cleavaged by hydroxyl radical (·OH),37-38 generated by the catalytic reaction of FL-Au@Pd39 and GQDs40-43 with H2O2 released from cells. The photocurrent further decreased with an increase in the concentration of the cells (curve e). This was due to the fact that the more cells in the modified electrode surface, the more ·OH will produce, thus, leading to the increase of DNA-bridge breakage. As a result, a certain amount of GQDs was cleaved off from the surface of the electrode, leading to lower photo-to-electron output. The cleavage of as-generated ·OH to single strand DNA was also verified by agarose gel electrophoresis images. As shown in Figure 4B, there was negligible effect to the single strand DNA sequences (lane a) by H2O2 (lane b). However, a weak band was obtained on the gel when there is coexistence of FL-Au@Pd NPs and H2O2 (lane c), owing to Au@Pd NPs possess higher peroxidase-like activity, which could serve as a promoter to convert H2O2 into ·OH. As a result, single strand DNA was damage.

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Figure 4. (A) Photocurrent responses of the stepwise fabrication of the PEC cyto-sensor; (B) Gel-electrophoresis analysis of the DNA cleavaged by as-generated ·OH. Lane a: DNA; Lane b: DNA upon H2O2 addition; Lane c: DNA upon Au@Pd NPs and H2O2 addition; Lane d: DNA upon GQDs, Au@Pd NPs and H2O2 addition

Similarly, after adding the GQDs, FL-Au@Pd NPs and H2O2 to the as-prepared single strand DNA sample, an obvious weak band was obtained (lane d), indicating the synergistic catalytic activity of GQDs and FL-Au@Pd NPs toward H2O2. The viability was characterized through microscopy imaging. Figure 5A and Figure 5B exhibited the bright-field optical image and fluorescence microscopy image of MCF-7 cell stained with Calcein AM (a living cell dyes) and propidium iodide (a dead cell dyes) after incubation with probe agents with cell for 1 h, respectively. From the figures we can see that the MCF-7 cells showed a good living morphology, indicating the low cytotoxicity of the GQDs/Apt/FL-Au@Pd toward the cells. Besides, longer incubation time was also investigated. As shown in Figure 5C and Figure 5D, the cells showed fusiform shape and a limited number of dead cells was observed, indicating the hybrids have no apparent effect on cancer cells. The efficiencies of cell capture and viability of captured cells was investigated. Figure

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5E,F exhibited the fluorescence microscopy image of the Pt-PWE surface-captured MCF-7 cells after 1 h (Figure 5E) and 3 h (Figure 5F) of incubation. The strong green fluorescence signals from the cells indicated that the cells were alive during the cytosensing processes.

Figure 5. Optic microscopy images (A,C)

and fluorescence microscopy images (B,D) of

MCF-7 cell stained with Calcein AM and propidium iodide after incubation with probe agents for 1 h (A,B) and 8 h (C,D); Fluorescence microscopy images (E,F) of calcein-AM and propidium iodide-stained MCF-7 cells after they were captured on the electrode surface for 1 h (E) and 3h (F).

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3.3. The kinetic analysis of the compound

Figure 6. (A) The concentration of TMB was 20 mM and the H2O2 concentration was varied. (B) Double reciprocal plots of activity of the composite with the concentration of one substrate fixed and the other varied.

To quantitatively investigate the catalytic activities of the composite material, the initial reaction rates versus the H2O2 concentrations were plotted in Figure 6. For the purpose of comparison, Km values of previously reported other enzyme mimics and HRP were listed in Table 1. The Km value of the GQDs/Apt/FL-Au@Pd for H2O2 was obviously lower than other peroxidase mimetics and natural enzyme. These results imply that GQDs/Apt/FL-Au@Pd have excellent peroxidase-like activity.

Table 1. Comparison of the Km of various enzyme mimics. Enzyme mimics

Pd NPs

HPR

graphene oxide

GQDs NPs

GQDs/Apt/FL-Au@Pd

Km [H2O2]/(mM) References

6.69 39

3.70 39

0.588 20

0.49 20

0.18 This work

3.4. Monitoring of the flux of H2O2 production by cells On the basis of the GQDs and FL-Au@Pd NPs synergistic catalytic action to convert H2O2 into ·OH and ·OH-damaged single strand DNA, the PEC and colorimetric 20


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cyto-sensor was applied for sensitive detection of H2O2 released from tumor cells

Figure 7. Typical PEC responses (A,B) and colorimetric response (C,D) of this µ-PEC/C incubated with different concentrations of MCF-7 cells. (B,D) Calibration curve for MCF-7 cells determination. Inset: colorimetric imaging of the µ-PEC/C incubated with different concentrations of MCF-7 cells, from a to f: 104, 105, 106, 5×107, 108, and 5×108 cells·mL-1, respectively.

under the optimal conditions (Figure S7 and Figure S8). Figure 7A shows the PEC signals measured at various concentrations of cells. As the concentration of the cells increased, the photocurrent intensity decreased gradually, indicating the enhancement of released H2O2. The calibration plot displayed a good linear relationship between photocurrent and the logarithm of the cells concentration in a range of 5×102 to 5×108 cells·mL-1 (Figure 7B). The photocurrent intensity of 6.16 µA was obtained after

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incubation of 1.0×107 cells·mL-1 (10 µL), corresponding to 6.76×10-15 mol of H2O2 being released from each cell which was estimated based on the calibration curve depicted in the Figure S9A,B and the number of cells used in the measurements were ~1.0×107 cells·mL-1. Besides, colorimetric analysis was adopted to perform visual detection of H2O2 released from cells. As shown in Figure 7C, the color of chromogenic zone turned from colorless to blue to a deep with increasing concentrations of cancer cells. Moreover, the cyto-sensor shows a linear relationship between the grey intensity and the logarithm of cancer cells concentration (Figure 7D). The grey intensity of 88.16 a.u. was obtained after incubation of 1.7×108 cells·mL-1 (10 µL), corresponding to 6.91×10-15 mol of H2O2 being released from each cell, which was estimated based on the calibration curve depicted in the Figure S9C,D. This two values agreed well with that reported previously (6.36-7.08×10-15 mol·cell-1).44 4. Conclusions In summary, we have successfully developed a µ-PEC/C cyto-device for sensitive and visual detection of H2O2 based on BL-ZnO modified Pt-PWE as sensor platform and GQDs/Apt/FL-Au@Pd as signal amplification probe. Because of the effective matching of energy levels between GQDs and BL-ZnO and the fast electron migration of the Pt-PWE, the µ-PEC/C showed amplified photocurrent intensity. However, the GQDs/Apt/FL-Au-Pd hybrids can convert H2O2 into ·OH radicals under the presence of H2O2, leading to the disassembly of GQDs/Apt/FL-Au@Pd hybrids. As a result, the PEC quenching of BL-ZnO and the grey intensity enhancement of

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TMB in each chromogenic zone could be induced, resulting in a new dual-potential PEC/C sensing approach for intracellular H2O2 detection. These results not only opened a different horizon for future PEC investigation through a judiciously engineered PEC nanosystem, but also offered a reliable and intuitionistic method in biosensing and clinical diagnosis. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21475052, 21207048).

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