Internal Light Source-Driven Photoelectrochemical 3D-rGO

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Internal Light Source-Driven Photoelectrochemical 3D-rGO/ Cellulose Device Based on Cascade DNA Amplification Strategy Integrating Target Analog Chain and DNA Mimic Enzyme Feifei Lan, Linlin Liang, Yan Zhang, Li Li, Na Ren, Mei Yan, Shenguang Ge, and Jinghua Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12338 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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ACS Applied Materials & Interfaces

Internal

Light

Source-Driven

Photoelectrochemical

3D-rGO/Cellulose Device Based on Cascade DNA Amplification Strategy Integrating Target Analog Chain and DNA Mimic Enzyme

Feifei Lan,†,‡ Linlin Liang,†,‡ Yan Zhang,†,‡ Li Li,†,‡ Na Ren,† Mei Yan,†,‡ Shenguang Ge,*,† Jinghua Yu,†,‡

†

Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan,

Shandong, 250022, P. R. China. ‡

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

Shandong, 250022, P. R. China.

*Corresponding author: Shenguang Ge E-mail: [email protected] Telephone: +86-531-82767161

ACS Paragon Plus Environment


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ABSTRACT: In this work, a chemiluminescence-driven collapsible greeting card-like photoelectrochemical lab-on-paper device (GPECD) with hollow channel was demonstrated, in which target-triggering cascade DNA amplification strategy was ingeniously introduced. The GPECD had the functions of reagents storage and signal collection, and the change of configuration could control fluidic path, reaction time and alterations in electrical connectivity. In addition, three-dimentional reduced graphene oxide affixed Au flower was in situ grown on paper cellulose fiber for achieving

excellent conductivity

and biocompatibility.

The cascade

DNA

amplification strategy referred to the cyclic formation of target analog chain and its trigger action to hybridization chain reaction (HCR), leading to the formation of numerous hemin/G-quadruplex DNA mimic enzyme with the presence of hemin. Subjected to the catalysis of hemin/G-quadruplex, the strong chemiluminiscence of luminol-H2O2 system was obtained, which then was used as internal light source to excite photoactive materials realizing the simplification of instrument. In this analyzing process, thrombin served as proof-of-concept, and the concentration of target was converted into the DNA signal output by the specific recognition of aptamer-protein and target analog chain recycling. The target analog chain was produced in quantity with the presence of target, which further triggered abundant HCR and introduced hemin/G-quadruplex into the system. The photocurrent signal was obtained after the nitrogen-doped carbon dots sensitized ZnO was stimulated by chemiluminescence. The proposed GPECD exhibited excellent specificity and sensitivity toward thrombin with a detection limit of 16.7 fM. This judiciously


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engineered GPECD paved a luciferous way for detecting other protein with trace amounts in bioanalysis and clinical biomedicine.

KEYWORDS: photoelectrochemical, lab-on-paper device, 3D-rGO, target analog chain, DNA mimic enzyme, thrombin



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1. INTRODUCTION Paper has become a research hot spot by virtue of the low cost and excellent biodegradability on actual occasion when the whole world is striving for energy-conserving

and

environment-protective.1-3

In

addition,

the

large

surface-to-volume rate caused by porosity is instrumental to the immobilization of functional materials, and the porosity of the paper could accelerate the diffusion of reagent.4 Moreover, the capillarity of the paper cellulosic fiber could drive the transport of fluids without external motivating force.5,6 Thereupon, portable and disposable paper-based devices have been widely used in various analysis fields such as personal inspection devices,7-9 biological medicine10 and environmental monitoring.11,12 Compared with traditional paper-based devices, greeting card-like photoelectrochemical lab-on-paper device (GPECD) with hollow channel enable the paper to be splendidly exploited by ingenious design. The framework of greeting card could enable different reactions to proceed simultaneously and can flexibly regulate the reaction time by time-coincidence reagents injection and GPECD folding. The design of hollow channel breaks through the barriers around low rates of convective mass transfer and significant nonspecific adsorption.13 Whereupon, the proposed novel GPECD with hollow channel is fabricated and employed taking thrombin (TRB) as a proof-of-concept target, which further promotes the prosperity of emerging paper-based devices with hollow channel. TRB, a crucial enzyme in blood coagulation, has been the object of attention in recent years on account of the crucial role in cardiovascular diseases, Alzheimer's


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disease, regulation of inflammation processes, anti-clotting therapeutics, and many other coagulation-related reactions.14-16 High-amount of TRB will give rise to thromboembolic diseases or even demise, while low-level of TRB will cause excessive hemorrhage. Hence, the novel biosensor that could realize rapid, sensitive and accurate TRB detection in clinical diagnosis is imperative. Compared the conventional

approaches,

such

as

electrochemilumin-escence,19 colorimetry20

electrochemistry,17

fluorescence,18

and surface plasmon resonance,21

paper-based photoelectrochemical biosensor with the merits of low cost, environmental protection and easy miniaturization has been a promising analytic method. Notwithstanding there are many detection methods for TRB in recent years, the sensitivity cannot meet the increasing demands in clinic medicine. To this end, the proposed GPECD was well designed on the substrate material and signal amplification strategy. Recently, carbon-based materials have been widely applied on account of high electron mobility22 and great thermal conductivity.23 The three-dimentional reduced graphene oxide (3D-rGO) was in-situ synthesized on the paper cellulose fiber (3D-rGO/cellulose) by green pollution free hydrothermal method recur to the surface functional groups and porous structure of paper.24-26 Differing from the all-purpose two-dimensional structure, the 3D-rGO greatly increased the specific surface area of the original, which was favorable for binding functional materials and the corrugated structure was preferred from the perspective of fast mass and electron transport kinetics.27 Hence, the promising 3D-rGO/cellulose with high



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electron-transport ability28 was selected as the substrate of the proposed biosensor, which laid a strong foundation for the fabrication of an ultrasensitive biosensor. In order to further improve the sensitivity of the GPECD, signal amplification strategy is undoubtedly a pregnant point of penetration. Numerous aptamers with highly specific and high-affinity have been selected against various kinds of target molecules including small organics, proteins, peptides and even viruses or cells.29 Contrasted with familiar antigen-antibody, aptamers have more meliority over storage, stability and reusability, and possess widespread availability for the vast majority of subsistent protein.30,31 Nevertheless, one target molecule generally could only trigger a target-aptamer binding event, which limits the signal collection and thus shows poor sensitivity. In view of this, target-induced cascade DNA amplification strategy based aptamer hairpin switch was employed our system. Moreover, it is well-known that exciting light source is an indispensable and decisive role in photoelectric chemical system. However, conventional physical light source frequently derived from supernumerary apparatus, which was contrary to the tenet of instrument miniaturization and operation simplification. In response to this challenge, the typical luminol-based chemiluminiscence emission serving as inner light source was introduced this system. The hydroxyl radical produced from H2O2 could catalyze the chemiluminescent reaction of luminol with the generation of chemiluminiscence. Additionally, the horseradish peroxidase serving as the catalyst of luminol-H2O2 system was superseded by burgeoning hemin/G-quadruplex. With the effect of target-induced

cascade

DNA

amplification

strategy,


the

amount

of

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hemin/G-quadruplex

introduced

on

the

electrode

directly

influenced

the

chemiluminiscence served as inner illuminant, which further gave rise to the change of photocurrent response and indirectly realized the accurate detection of target analyte.

Herein, an ingenious 3D-rGO/cellulose based GPECD was constructed via the target-triggered cascade DNA amplification. On the one hand, the Au modified 3D-rGO with superior conductivity was grown on the paper cellulosic fiber of test tab (Au@3D-rGO/cellulose). Subsequently the photoactive material, ZnO with broad band gap was immobilized on the Au@3D-rGO/cellulose surface with the aid of 4-aminothiophenol. Afterwards, the emerging nitrogen-doped carbon Dots (N-Cdots) with narrow band gap were anchored on the ZnO modified Au@3D-rGO/cellulose forming the photoelectric composite materials with different absorption ranges due to their different band gaps. After the capture probe bound on the N-Cdots via amido bond, the as-prepared test tab was put away for following assay. On the other hand, target analog chain (TAC) was produced with the presence of target. The released target complex was available for initiating next cycle, which was promising for signal amplification. The TAC could not only bind with capture probe but also trigger the hybridization chain reaction (HCR).The designed hairpin probe in HCR could form the structure of hemin/G-quadruplex with the help of hemin. The numerous mimic enzyme hemin/G-quadruplex were drawn into the test tab once the HCR was triggered by TAC, which offered a predominant catalytic environment for the luminol-based chemiluminescne reaction with p-iodophenol served as enhancer. The variation of



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chemiluminescence served inner excitation source further influence the photocurrent signal output of the N-Cdots sensitized ZnO in proposed GPECD. The hairpin probe used in this system can be easily designed for other proteins by changing the corresponding aptamer. Therefor the low-cost and disposable GPECD provide a versatile tool in ultrasensitive detecting multiple biomolecules in bioanalysis and clinical biomedicine.

2. EXPERIMENTAL SECTION 2.1. Design and Fabrication of the GPECD. The GPECD was prepared referred to our previous work32,

33

with slight modification, and the detailed

fabrication procedure was represented in the Supporting Information (Figure S1 and S2). The parameter setting about wax-printing was shown in Table S1. The GPECD was comprised of pretreatment tab, test tab, auxiliary tab and circuit boards. As shown in Scheme 1A, the reaction zones I and II in pretreatment tab were designed for storing with reagents and offering zones for the formation of TAC and hemin/G-quadruplex respectively. Substrate materials and photoelectric materials were modified on another side of text tab with carbon working electrode. The as-prepared test tab and auxiliary tab with screen-printed reference electrode and counter electrode were embedded in the folded pretreatment tab, then further assay and measurement were proceed while the GPECD were assembled with two self-made circuit boards and clapped with binder clip (Scheme 1B and Scheme S1A). In this assay, the reacted reagents were drained to the reservoir for further assay once the GPECD was pressed together with the assistance of hollow channel and


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microfluidic channel. The overlapped structures were presented in Scheme 1C. Particularly, reaction zones I, II and microfluidic channel were partially deposited solid wax and presented semi-hydrophilic (Scheme 1D), which was aim to avoid the exosmosis and pollution of reagents with different zones and provide guarantee for different reactions taking place simultaneously. The real size of components in GPECD was presented in Scheme S1B. 2.2. Preparation of Au@3D-rGO/cellulose. The fabrication process of Au@3D-rGO/cellulose mainly includes two parts (schematic representation in Figure S3). To begin with, the 3D-rGO/cellulose was obtained by a parallel process used in the synthesis of 2D-rGO/cellulose (details in Supporting Information) in which vacuum freeze drying was introduced. Briefly, the obtained 2D-rGO/cellulose by washing with ultrapure water was placed in vacuum freezer dryer for 8 h so as to form the 3D-rGO on cellulose. And then the Au flower was growth on the 3D-rGO/cellulose through a direct chemical reduction of HAuCl4 by ascorbic acid according to previous reported literature.34 The 60 µL of as-prepared Au NPs seeds solution35 (details in Supporting Information) was added to the obtained 3D-rGO/cellulose and kept for 1 h at room temperature, repeating five times in order to optimize the surface immobilization of Au nanoparticles seeds on cellulose fibers. After the unbound the Au NPs seeds washed off with ultrapure water, taking care that the solution of HAuCl4 (10 mM) and ascorbic acid solution (100 mM) were mixed and dropped on the paper work zone quickly. After reaction of 15 min at room temperature, the resulting Au@3D-rGO/cellulose was washed with ultrapure water



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thoroughly and dried at room temperature, the resulting Au@3D-rGO/cellulose was washed with ultrapure water thoroughly and dried at room temperature.

Scheme 1. (A) Schematic representation, size and shape of this GPECD; (B) Schematic representation of the process of fabrication for GPECD (circuit pads a, b, c, d for the connection to reference electrode, counter electrode, working electrode and analyzer through alligator clip); (C) Exploded view: stacking of several patterned layers of paper for fabricating the GPECD; (D) Schematic illustration of paper fiber with different permeation of wax.

2.3. Assay Protocol of the GPECD. To begin with, the solutions of HP1, HP2, H1, H2 (details in Supporting Information) were heated to 95 °C for 10 min and cooled to 4 °C by using a PTC-200 thermal cycler (MJ Research Inc., Waltham, MA )


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to form the stem-loop DNA structure. In this study, the stock solutions of HP1, HP2 were diluted in 60 µL of buffer solution (10 mM Tris-HCl and 50 mM MgCl2, pH 8.0) contained 20 U T7 exonuclease (T7Exo) to the final concentration of 1.0 µM and 3.0 µM respectively. After the 50 µL of mixture solution was dropped on the reaction zone I by toppette pipettor, 5.0 µL of TRB sample solution with certain concentration was pumped into the above solution quickly for reaction with 100 min. After 40 min, the stock solutions of H1, H2 were diluted in 60 µL of buffer solution containing 2.0 µM of hemin and 300 mM of K+ for the final concentrations of 10 µM. Then 50 µL of mixed solution was pumped into the reaction zone II for incubating with 60 min to form hemin/G-quadruplex. After the preprocessed test tab loaded with capture probe (details shown in Supporting Information) was embedded in the pretreatment tab, GPECD was closed and maintained at room temperature for 30 min. Thereafter the biosensor was carefully rinsed with buffer solution to remove the physically absorbed materials, the prepared auxiliary tab with reference electrode and counter electrode was embedded in the corresponding embedded region (Scheme 1A), then which was covered via the designed circuit boards and clamped via binder clips. Next, the GPECD was connected to the electrochemical workstation. Ultimately, 100 µL of phosphate-buffered solution containing luminol (0.5 mM), H2O2 (5.0 mM) and p-iodophenol (0.5 mM) was injected into the GPECD via the hole in circuit board (Figure S4), followed by the photocurrent measurement. 3. RESULTS AND DISCUSSION



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Scheme 2. (A) Schematic illustration of cycle production of target analog chain and its application in system; (B) Photoelectric chemical mechanism of GPECD under the internal light source. Principle of internal light source-driven GPECD.

3.1. Principle of Internal Light Source-Driven GPECD. The principle of production for TAC was illustrated in Scheme 2A. With the presence of target, the hairpin DNA probe (HP1) with aptamer of TRB (a, β, γ, δ) would specifically combine with the TRB to form HP1@TRB structure, and then whose exposed segment

hybridized

with

the

designed

hairpin

DNA

probe

labelled

dibenzocyclooctyne (DBCO) at 5′-end (HP2). The formed HP1@TRB@HP2 was digested by T7Exo causing the liberated HP1@TRB for new circulation and the region of ζ, ε*, γ* in HP2 for triggering the following HCR. The amount of the free signal-chain domain marked ζ, ε*, γ* has intimate connection with the target, whereupon it was defined as TAC (target analog chain). Additionally it is notable that the T7Exo could specifically cleave duplex DNA from the recessed 5′-end, which the reason why HP1 and HP2 consist of a protruding single-stranded fragment at their 5′ termini.36 The TAC was successfully fastened onto the as-prepared test tab as initiator of HCR by virtue of metal-catalyst free click chemistry reaction with


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DBCO-containing TAC and an N3-containing capture probe, which averted the interference from coexisting biomolecules and groups.37,38 Subsequently, the luminol-H2O2 system yield a chemiluminescence emission with the catalysis of hemin/G-quadruplex. Driven by the chemiluminescence emission, as shown in Scheme 2B, N-Cdots were firstly photo-excited to produce an electron (e-)-hole (h+) pair. Then electrons in conduction band transferred to conduction band of ZnO with broad band gap. Followed by the injection of the conduction-band electrons into the carbon working electrode through Au@3D-rGO/cellulose, photocurrent signal was yielded. Meanwhile, H2O2 served as oxygenant in luminol-based system also served as the electron donor that provided electrons to the hole (h+). 3.2. Materials Characterization. Paper with rough surface and porous architecture (Figure 1A and B) was selected as the substrate material in our system on account of its quick chemical transport rates, adsorbability, flexibility, low cost. The conductivity of substrate material plays vital role in biosensors analysis. Hence, conductive rGO was synthesized on the cellulose fibers. As shown in Figure 1C, the 2D-rGO was assembled on the paper via a hydrothermal process and could connect well with the adjacent rGO (showed in the ellipse). Enlarged view Figure 1D revealed that rGO coated on the cellulose fibers presented flake structure with slight wrinkles. The coating of compact 2D-rGO made the paper resistant to the inter-fiber sliding due to the mechanical locking, which immensely improved the mechanical properties of the cellulose paper.



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Figure 1. SEM images for morphologies of the paper before and after growth of 2D-rGO. (A) Surface of bare paper; (B) Cross-section of bare paper; (C) 2D-rGO/cellulose; (D) Enlarged 2D-rGO/celluloses.

The hydrothermally produced 3D-rGO undergone freeze-dry was obtained. The corresponding SEM (Figure 2A-D) revealed that the 3D-rGO had a looser structure with much thinner connection walls than the 2D-rGO. The richly wrinkled structure could further improve the active area of rGO layers for the growth of Au flower. Such architecture attributed to the crisscross fiber network and microporous structure of the paper, which not only facilitated the electron transport throughout the paper but also refrained from the aggregation of rGO sheets in the pores of paper. Raman spectra in (Figure S5) also indicated that the rGO layers was derived from GO layers by chemical reduction, which were accordant with the previous report.39 As shown in Figure 2E, the Au flower with the average diameter around 400 nm was distributed uniformly in the wrinkle of 3D-rGO, which further improve the electrical conductivity of the substrate material and offer suitable microenvironment for bio-experiments.


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Compared

with

the

3D-rGO

coated

cellulose

fiber,

the

conductivity

Au@3D-rGO/cellulose was tremendously increased, which were testified by the electrochemical impedance spectroscopy characterization. As shown in Figure S6, Au@3D-rGO/cellulose (curve c) possessed the smaller electron-transfer resistance than 3D-rGO/cellulose (curve b), which was accordant with the CV response in Figure 4A (curve b and c). The element peaks of C, O, Au in energy dispersive spectroscopy (EDS) analysis (Figure S7A) confirmed the formation of Au flower on paper. Moreover, the EDS mapping images, as shown in Figure 2F, were obtained to analyze the elemental distribution. The images revealed that the Au flowers were evenly distributed throughout the paper, which were consistent with the corresponding SEM images and the size distribution of Au flower shown in Figure S7B.

Figure 2. SEM images of the 3D-rGO/cellulose (A-D), Au@3D-rGO/cellulose (E); EDS mapping images of gold element, carbon and oxygen of the Au@3D-rGO/cellulose (F).



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The synthesized ZnO was astroid in shape with average size of 2 µm, as revealed by SEM (Figure 3A). The astroid ZnO architecture consisted of plentiful petal-like nanostructure. The protrusive petals not only offered numerous active sites for quantum dots but also were beneficial to the electron transfer from photoelectric material to Au@3D-rGO/celluloses. Furthermore, the crystal structure of ZnO was analyzed by X-ray diffraction (XRD). As shown in Figure3B, the obvious diffraction peaks appeared at 31.94, 34.62, 36.40, 47.04, 56.52, 62.86, 66.29, 68.17, 69.24 ° assigned to the 100, 002, 101, 102, 110, 103, 200, 112 and 201 planes of the wurtzite structure ZnO with lattice constants a=3.249 and c=5.206 Å, which were coincidently matched with the values in the standard card (JCPDS File No. 36-1451). The diffraction peaks were high and narrow, suggesting that the ZnO had a high degree of crystallinity. No diffraction peaks of other impurities were observed implying pure ZnO structures were synthesized through this fast and simple hydrothermal process.

Figure 3. SEM (A) and XRD pattern (B) of the ZnO nanostructure synthesized by hydrothermal process; (C) TEM and of the N-C dots; (D) XPS spectra of the obtained N-Cdots; (E) XPS C 1s analysis of the N-Cdots; (F) XPS N 1s analysis of the N-Cdots.


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The N-Cdots with average diameter about 6 nm, shown in Figure 3C, are well separated from each other and monodispersed without apparent aggregation. Moreover, X-ray photoelectron spectroscopy (XPS) and fourier transformed infrared (FT-IR) were carried out for analyzing the components and surface functional groups of the as-obtained N-Cdots. The triumphant doping of nitrogen element was testified by the XPS in which the distinct peak at 399 eV derived from the N 1s binding energy of the C-N bond. Meanwhile the peaks of C 1s (286 eV) and O 1s (532 eV) presented in the wide spectrum of N-Cdots (Figure 3D). The high-resolved C 1s XPS spectrum (Figure 3E ) was deconvoluted into three peaks at 284.8, 288.2 and 289.6 eV, which represent C 1s states in C-C/C=C, C-O/C-N and C=O/ C=N respectively. In the N1s XPS spectra (Figure 3F), the fitted peaks at 398.8 eV and 400.5 eV were assigned to pyridinic N (C-N-C), and graphitic N (N-C3) or pyrrolic-N (C2-N-H).40,41 The FT-IR of N-Cdots (Figure S8A) showed the absorption peak at around 792 cm−1 attributed to the C-O stretching vibrations, the peak centered at 1097 cm−1 assigned to C-N and C-OH stretching vibration, the peak at about 1258 cm−1 attributed to N-H stretching vibration, the peak at 1656 cm−1 attributed to C=O stretching vibration, the peak at 2928 cm−1 attributed to C-H stretching vibration, the broad peak centered at 3431 cm−1 attributed to O-H and N-H stretching vibration. The peaks of carbonyl and hydroxyl with strong intensity demonstrated that the N-Cdots possessed excellent water solubility and hydrophilic property, which benefits for further coupling in experiment. The surface components and surface functional groups of the N-Cdots proved by the FT-IR were consistent with the corresponding XPS spectra.



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The admirable optical properties of the N-Cdots were investigated by the ultraviolet-visible (UV-vis) absorption and fluorescence (FL) spectra (Figure S8B). The UV-vis spectrum of the as-obtained N-Cdots showed an absorption peak at 275 nm, which was assigned to the π-π* transition of aromatic sp2 domains and demonstrated the existence of aromatic cycle. FL spectra were measured under the different excitation wavelength, and the excitation-dependent emission was observed. In addition, the N-Cdots showed strong photoluminescence in the range of 400-600 nm, the maximum emission peak was observed at 452 nm with excitation of 360 nm. The above-mentioned results are consistent with recent report on nitrogen-doped carbon nanoparticles.43, 44 3.3. Photoelectrochemical Characterization. In PEC biosensor, the property of photoelectric materials was vital to the sensitive detection for targets and the electron transfer mechanism was shown in Scheme 2B. Thus the photoelectric properties of ZnO and N-Cdots were investigated by photocurrent measurement (Figure S9). The N-Cdots obtained by the microwave- assisted method was chemically conjugated on the astroid ZnO forming N-Cdot/ZnO. The pristine ZnO could only harvest ultraviolet light, leading to little photocurrent conversion efficient. Compared with the ZnO, the N-Cdot/ZnO could generate stronger photoelectrochemical response (curves d and a). The N-Cdot increased the absorption range to longer-wavelength light, which could facilitate the injection of photogenerated electrons from N-Cdots to ZnO. In addition, the result that N-Cdot functionalized ZnO had a much enhanced photocurrent


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response compared to undoped Cdots was obtained (curves d and c), which manifested the doping of nitrogen had a role in promoting electron transport.

Figure 4. (A ) CV responses of (a) bare paper, (b) 3D-rGO/cellulose, (c) Au@3D-rGO/cellulose, (d) ZnO modified Au@3D-rGO/cellulose, (e) step d bound with N-Cdots, (f) step e attached with capture probe, (g) step f jointed with TAC by click chemistry, (h) step g hybridize with H1, H2; (B) Photocurrent responses of the GPECD toward various concentrations of TRB: (a-j) 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 pM; (C) Typical PEC responses. Insert: calibration curve for TRB determination; (D) Selectivity evaluation of the GPECD detection of thrombin (100 pM) against the interference proteins (1000 pM, n= 3).



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The triumphant layer-by-layer assembly of the GPECD was also allimportant to the sensitive detection of targets, so the cyclic voltammetry (CV) was conducted in 5 mM [Fe(CN)6]3-/4- solution containing 0.1 M KCl at 100 mV/s scan rate. As could be seen in Figure 4A, the bare paper exhibited a well-defined redox peak of [Fe(CN)6]3-/4- (curve a). The redox peak current increased as the result of the growth of 3D-rGO on the surface of cellulose (curve b), indicating the superior conductivity of 3D-rGO. Amazingly, the redox peak current had an obvious increase after the Au flower grown on the wrinkle of the 3D-rGO (curve c), which resulted from that conductive Au could effectively accelerate the electron transfer. Subsequently, the CV response decreased sequentially (curves d and e) when astroid ZnO and N-Cdots were modified on the surface of the Au@3D-rGO/cellulose, testifying that the assembly of photoelectric material and sensitizer hindered the transmission of electrons. After the capture probe and TAC were bound onto the as-obtained electrode, obvious decrease in redox peak current was observed (curves f and g), which was because the negative charged phosphate backbone of capture probe and TAC could be able to impede the electron transmission from solution to interface of electrode. Incubated with H1 and H2, the redox peak current further decreased because of the negative charged phosphate backbone and steric hindrance of hemin/G-quadruplex. The above mentioned results manifested the successful fabricated process of the GPECD. 3.4. Analytical Performance. The photocurrent signal of the GPECD depended on the fixation amount of hemin/G-quadruplex by the HCR at the trigger of TAC that had direct relation with the concentration of TRB. Under the optimal conditions


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(Figure S10), the response signals for targets with various concentrations were recorded in Figure 4B. The photocurrent intensity increased gradually with the elevated concentration of TRB (Figure 4C). The more target TRB presented, the more TAC could be generated. The presence of more TAC could trigger the more hybridization chain reaction, and more hemin/G-quadruplex serving as catalyst of chemiluminescence reaction was introduced into the GPECD. Thus, the enhancing photocurrent response was recorded under the condition contained luminol and H2O2 after the stronger chemiluminiscence was obtained. A calibration curve was made by plotting photocurrent response versus the logarithm of the TRB concentration, with presented a good linear relationship in a range of 50 fM to 1000 pM. The equation of the calibration curve was I = 323.160 + 151.241 logcTRB, with a correlation coefficient of R = 0.994. Additionally, the limit of detection was estimated to be 16.7 fM (based on 3σ), which was lower than that in some previous reports (Table S2). TAC generated from target-analog recycling amplification strategy and double helices of DNA equipped with abundant hemin/G-quadruplex were ingeniously integrated into in this biosensor, which were the reason why the GPECD had such low detection limit. The specificity, stability and reproducibility of the GPECD also were especially important in the practical applications. The selectivity of the designed GPECD for TRB detection was examined by challenging it with IgG, CEA, BSA, TRB as well as a mixture of them and the results were displayed in Figure 4D. From this investigation, we could see that the presence of the interference proteins at 1000 pM exhibited an



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almost neglectable response. However, the photocurrent response increased obviously after the incubation of TRB and its mixture contained disturbed non-targets. The above mentioned results demonstrated that the proposed GPECD possessed an excellent specificity in the assay of the TRB, which principally attributed to the highly specific interactions between the aptamer and related target. The stability of the developed GPECD also was evaluated by detecting the photocurrent response of the GPECD with different storage time at 4 °C (Figure S11). Notwithstanding the photocurrent response decreased progressively with the extension of storage time, the response retained 90.63% of the initial photocurrent signal after four weeks with negligible loss, which demonstrated the prepared GPECD had an acceptable stability. Moreover, the reproducibility of the GPECD was examined with two concentrations (10 and 500 pM). The relative standard deviations (RSD) of intra-assay respectively (n=6) were 4.7% and 4.1%. Meanwhile, the interassay RSDs of 5.2% and 4.8% were obtained by measuring the targets of the same concentrations with six devices fabricated independently under identical experimental conditions. These results indicated satisfactory reproducibility of the proposed GPECD. 3.5. Real Sample Analysis. To further appraise real applicability of the GPECD, a series of different concentrations of TRB were detected by the standard addition method. The background signal originate from the serum sample was took out the quantitative assay. As exhibited in Table S3, we could discover that the recovery (98.16% to 102.14%) and RSD (2.1% to 4.2%) were satisfactory, which clearly


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declared the designed GPECD possessed great potential for the detection TRB in real biological samples. 4. CONCLUSIONS In summary, we proposed a foldable GPECD with hollow channel for ultrasensitive TRB detection based on the cascade DNA amplification strategy. In this system, the ingenious design of GPECD with hollow channels not only could expediently regulate timing for reaction progress but also facilitate the fluid transport recurring to capillary flow of the cellulose fiber. In addition, the formation of 3D-rGO/cellulose with excellent conductivity and accrescent surface area on account of the abundant plicated structure offered a promising platform for the construction of photoelectrochemistry biosensors. Moreover, the T7Exo assisted DNA digestion process could produce abundant cleaved TAC with the initiators of HCR, which acted as the primers to initiate the hybridization reaction between H1 and H2 contained hemin/G-quadruplex. The introduction of mimic enzyme promoted the luminol-based chemiluminescent reaction, which enhanced the inner light source for the proposed system and thus irradiated the N-Cdots covalently conjugated ZnO accompanied with conspicuous enhancement of the photocurrent response. Therefor this inexpensive, disposable and sensitive GPECD furnish a feasible and robust tool for the detection of biomolecules with trace amounts in bio-analysis and clinical biomedicine. ASSOCIATED CONTENT Supporting Information



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Materials, apparatus, design and characterization of the GPECD, pretreatment of the text tab, schematic representation of the Au@3D-rGO/cellulose, GPECD assembled with circuit boards, raman spectra of GO and 3D-rGO, EIS characterization, EDS of Au@3D-rGO/cellulose, size distributions of Au folwer, FTIR spectrum and UV-vis absorption spectrum and fluorescence spectra of N-Cdots, photocurrent responses characterization, optimization of assay conditions, comparison of different biosensors for the detection of thrombin, assay results of TRB in human serum sample. The Supporting Information is available free of charge

via the Internet at

http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel: +86-531-82767161; Fax: +86-531-82765969; *E-mail address: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

This work was financially supported by National Natural Science Foundation of China (21575051, 21775055), Key Research and Development Program of Shandong Province, China (2016GGX102035), and Young Taishan Scholars (tsqn20161036). Furthermore, we are grateful for Haiyun Liu for his guidance on language.


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TOC 1058x554mm (72 x 72 DPI)


Internal Light Source-Driven Photoelectrochemical 3D-rGO

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