Colorimetric and Electrochemiluminecence Dual Mode Sensing of

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Colorimetric and Electrochemiluminecence Dual Mode Sensing of Lead Ion Based on Integrated Lab-on-Paper Device Jinmeng Xu, Yan Zhang, Li Li, Qingkun Kong, Lina Zhang, Shenguang Ge, and Jinghua Yu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18542 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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ACS Applied Materials & Interfaces

Colorimetric and Electrochemiluminecence Dual Mode Sensing of Lead Ion Based on Integrated Lab-on-Paper Device Jinmeng Xu,†,# Yan Zhang,†,# Li Li,† Qingkun Kong,† Lina Zhang,‡ Shenguang Ge,† Jinghua Yu*,†

†

School of Chemistry and Chemical Engineering, Institute for Advanced

Interdisciplinary Research, University of Jinan, Jinan 250022, P.R. China ‡

Shandong Provincial Key Laboratory of Preparation and Measurement of Building

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

∗

Corresponding author: Jinghua Yu

E-mail address: [email protected] Tel: +86-531-82767161; Fax: +86-531-82765956.

KEYWORDS: lab-on-paper device; visual prediction; electrochemiluminecence quantification; metal ion; DNAzyme; PdAu nanocrystals

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ABSTRACT: A highly selective two-point separation strategy was designed based on a cross-like all-in-one lab-on-paper analytical device. The stable and cleavable enzyme-coated reduced graphene oxide (rGO)-PdAu probe was fabricated as the signal reporter to enable visualization and electrochemiluminecence (ECL) dual mode sensing of Pb2+. Concretely, the experimental workflow consists of the following process, (i) fabrication of the lab-on-paper device and growth of Au NPs on ECL detection zone, (ii) immobilization of Pb2+-specific DNAzyme, (iii) hybridization between DNAzyme and rGO-PdAu-glucose oxidase (GOx) labeled oligonucleotide to form double-stranded DNA. Upon addition of Pb2+ into the prepared system, the double helix structure of DNA was destroyed, resulting in the release of cleaved rGO-PdAu-GOx probe to visualization bar to promote the effective oxidation and color change of 3, 3’, 5, 5’-tetramethylbenzidine. As a consequence, the color change can be recognized by naked eye, meanwhile GOx on uncleaved signal probe can oxidize glucose along with H2O2 production. As coreaction reagent for luminol ECL system, the concentration of H2O2 is proportional to the ECL intensity, which constitutes a new mechanism for colorimetric and ECL dual mode to detect Pb2+. With the method developed here, the concentration of Pb2+ could be easily determined by the naked eye within a linear range from 5 to 2000 nM as well as by monitoring the decreased ECL intensity of luminol in a linear range of 0.5 to 2000 nM. This work not only constructs a simple and versatile platform for on-site visible monitoring of Pb2+ in tap water and river water, but also furnishes a strategy for designing dual mode sensing toward different heavy metal ions based on specific DNAzyme in the fields of environmental monitoring related technologies.

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1. INTRODUCTION The development of simple and efficient device to identify specific toxic metal ions has been one intense issue in chemical and biological domain.1-2 Among various metal ions, lead ion (Pb2+) is included in the range of strong contaminants largely due to its perdurability, toxicity, and undegradable characteristic.3 It has a strong potential toxicity for many life tissue and long-term durability in the environment.4 Generally, the Pb2+ content in blood, by law in United States Environmental Protection Agency, is considered safe when it is ≤ 480 nM, and such a standard has been widely used in Asia and European Union.5 Great concern in Pb2+ detection also closely relates to the fact that this value should have to be precisely controlled to ensure health.6-8 As a consequence, it is of great significance to detect Pb2+ in water samples sensitively and selectively. A variety of methods, such as spectrophotometry,9 fluorescence,10 atomic absorption spectrum,11 and atomic emission spectrometry,12 have been extensively used for Pb2+ detection. Unfortunately these methods usually require high costs, time-consuming process, and specific experts to run the systems. Consequently, it is necessary to explore simple, portable, and inexpensive devices for rapid and on-site Pb2+ determination as well as effectively avoid the environmental contamination and Pb2+

intoxication

by

controlling

the

Pb2+

release.13-14

Colorimetric

and

electrochemiluminescence (ECL) technologies have emerged as two of the most prominent techniques of detecting heavy metal ions. Naked-eye colorimetric strategy, as a convenient analyte detection method, has offered an attractive alternative because of its easily monitored color changes without the need of any external sophisticated instruments and trained technician.15-16 ECL technology as a powerful analysis tool generated by electrochemical reactions between electrogenerated species,17 is competitive with conventional assays in Pb2+ determination due to its high sensitivity, excellent controllability and low background.18 As a consequence, optical probes with colorimetric and ECL dual modes are highly desired because they not only enable the semiquantitative visualization of Pb2+ simply with bare eyes prior to precise 3



determination but also validate a quantitative ECL assay in a simple and rapid feature.19 If the concentration of Pb2+ is within the allowable range prognosis by prediction, simple visualization could eliminate or minimize most costs associated with instrumentation and operation in ECL detection and thus can make on-site and real-time detection easier.20 Recently, the nanomaterials, such as reduced graphene oxides (rGO)21 and PdAu nanoparticles,22 have been studied as electrochemical signal amplification materials to construct integrated platform. PdAu bimetallic nanoparticles represent one class of attractive materials due to their multifunctionality such as catalytic, magnetic, sensing and optical properties.23 Particle type nanomaterials easily suffer from the aggregation, resulting in the activity degradation. The uniform distribution of PdAu nanoparticles on some supports can effectively address this problem.24 RGO, a 2D sp2-hybridized honeycomb-like carbon matrix, has become a remarkable supporting material for preparation of graphene-based nanocomposites due to their large planar surface, excellent electronic conductivity, high chemical stability, and mechanical strength for the determination of heavy metal ions and nocuous analytes.25,26 Various rGO-supported metal nanomaterials have been synthesized to construct many versatile platforms for heavy metal ions sensing with enhanced sensitivity and selectivity.27 Furthermore, enzyme-based signal amplification strategy has become the most popular technique in actual analysis due to the preponderances of high selectivity and specificity toward the substrates.28-29 So far, considerable efforts have been made to develop enzyme-based signal probes.30-31 Glucose-oxidase (GOx) is widely used in the field of food and medicine owing to the properties of low-cost and stable because it can specifically catalyze the oxidation of glucose with the cosubstrate oxygen accompanied with the production of gluconate and hydrogen peroxide.32 In current work, rGO-supported PdAu bimetallic nanoparticles coated with GOx was used as cleavable nanoprobes to enable the two-point separation method of visualization and precision detection of Pb2+ in water samples. Paper as an important invention of ancient China is the long-term accumulation of experience of the Chinese laboring people and the crystallization of human wisdom. 4


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Microfluidic lab-on-paper analytical chips have continued to develop at an exponential rate33-35 since the Whitesides’ group introduced the first example of the field due to its intrinsic characteristics.36,37 As a consequence, versatile lab-on-paper devices have been proposed for point-of-need precaution and detection.38-41 In this regard, a cross-shaped lab-on-paper device was designed, which consists of detection tab, channel tab, reference tab, and auxiliary tabs. Two black auxiliary tabs could be used to build a simulative dark environment via simple folding. The resulting lab-on-paper device enable novel dual mode sensing of Pb2+ in water samples synchronously. Here, we aim to developing an optical probe for colorimetric and ECL dual mode to Pb2+ detection based on crossed-like lab-on-paper device, where they not only enable the visualization of Pb2+ with naked eye, but also validate a quantitative ECL assay in real samples. As a whole, we design a cross-shaped origami-based multifunctional equipment. The working principle for such the assay is mainly on the basis of the specific biorecognition between the target and Pb2+-dependent DNAzyme (P1). Once lead (II) ion was introduced into the system, Pb2+-dependent DNAzyme will be activated. The activated DNAzyme would catalyze the ribonucleotide (rA) site as well as cleave the substrate strand (P2). As the cleaved strand driven down the channel by wicking properties to the visualization bar loaded with H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB), color variance can be signaled to estimate Pb2+ content. Furthermore, the uncleaved probe can participate the precise ECL determination. The more P1 cleaved, the deeper color of the TMB oxidation product and the smaller ECL signal would be obtained. As a consequence, the dual mode sensing of lead ion based on proposed lab-on-paper device can thus be simply completed with the changes in the color and the ECL intensity of luminol, respectively. The original lab-on-paper methods elaborated above is rapid and technically simple, which holds a tremendous prospect for integration inspection and on-site analysis of heavy metal ions in various researches.

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2. EXPERIMENTAL SECTION 2.1. Design of Functional Tabs. Enlightened by the fascinating merits of the lab-on-paper device, an origami dual mode device with cross-shaped structure was elaborately designed. As shown in Scheme 2A&B, the unique device was comprised of one detection tab (red) in the center position surrounded by four folding tabs: one channel tab (blue), one reference tab (green) and two auxiliary tabs (black). A detection zone (6.5 mm in diameter) for ECL quantification and a visualization bar (15 mm in length and 2.0 mm in width) for colorimetric prediction were patterned on the detection tab. To achieve the automatic flow of liquid, a circular region (6.0 mm in diameter) and a rectangular region (15 mm in length and 2.0 mm in width) connected by branch channels (3.0 mm in length and 2.0 mm in width) were patterned on the channel tab. After specific folding, ECL detection zone and visualization bar can be contacted with branch channels totally (Scheme 2D) to permit fluid flow and visual prediction. Via rectangular region (3.0 mm×3.0 mm) on the reference tab was sealed by silver, which was covered by the Au NPs layer to make both side of the sample tab conductive on the reverse side (Scheme 2B). Carbon working electrode will be connected with Ag/AgCl reference electrode and carbon counter electrode to realize precise ECL detection (Scheme 2C), enabling novel dual mode sensing for Pb2+. The function of the auxiliary tabs was based on its own black wax pattern, which could combine the channel tab and the reference tab to create a dark environment after folding to avoid the interference from external natural light. More detailed procedure on the fabrication of functional tabs could be found in Supplemental Material (Figure S1&2). 2.2. Synthesis of P2-rGO-PdAu-GOx Complex. Initially, the PdAu bimetallic nanocrystals were prepared via a facile seeded growth approach by employing Pd nanocubes as seeds according to the previously reported literature with slight modification.42 Briefly, poly (vinyl pyrrolidone) (PVP, 105 mg), L-ascorbic acid (L-AA, 60 mg), KBr (300 mg), Na2PdCl4 (57 mg) was suspended in 10 mL ultrapure water in a round bottomed flask. The reaction system was put in a 80 °C oil bath 6


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under magnetic stirring for 3 h and cooled down to room temperature. The Pd sample was washed with ethanol and ultrapure water by repeating the centrifugation and re-dispersion procedure several times to remove the excess bromide ions before being used as seeds. Afterwards, 1.0 mL Pd nanocubes aqueous suspension and 5.0 mL of an aqueous solution containing PVP (5 mg) and L-AA (3 mg) were added into a 20 mL vial. The mixture was heated at 95 °C for 20 min under magnetic stirring. Meanwhile, HAuCl4 aqueous solution was injected into the vial using a pipette. The reaction mixture was continued with heating at 95 °C for 15 min, and then cooled down to room temperature. After washing with ethanol and water, PdAu bimetallic nanocrystals were kept at 4 °C for further use. The

rGO-supported

PdAu

bimetallic

nanocrystals

coated

with

GOx

(rGO-PdAu-GOx) was synthesized according to the published method43 utilizing ethylene imine polymer (PEI) as cross-linking agent. First, 2.5 mL of GO dispersion (2.0 mg⋅mL-1) was dissolved in 7.5 mL ultrapure water. Then, 10 mL PEI (5%, w/w) and 100 µL NH3⋅H2O were added into the GO solution under magnetic stirring for 30 min. Next, the mixture was allowed in an oil bath for 12 h at 60 °C. In the end, 1.0 mL of the proposed solution was reacted with 300 µL PdAu bimetallic nanocrystals in an oil bath for 12 h to synthesize the rGO-PdAu nanocomposites. The obtained rGO-PdAu nanocomposites were centrifugated out, washed with ultrapure water several times for further purification. Subsequently, 10 mg portion of rGO-PdAu nanocomposites was dissolved thoroughly in 10 mL of Tris-HCl solution (10 mM, pH 8.0) by sonication. The rGO-PdAu-GOx were freshly prepared by adding GOx (100 µg⋅mL-1) into 1.0 mL rGO-PdAu solution under homogeneous mixing for 2 h. After that, the obtained rGO-PdAu-GOx nanomaterials was redispersed in blocking solution by continuous sonication for another 2 h. Excessive reagents were removed by centrifuging at 8000 rpm for 10 min. rGO-PdAu-GOx is readily functionalized with throated P2 via the well-known gold–sulfur chemistry. Conjugates of oligonucleotide P2-rGO-PdAu-GOx were synthesized following the protocol with a slight modification. Briefly, thiol-modified P2 (10 µL, 10 mM) was activated with 1.0 mL of 0.5 M Tris-acetate buffer (pH 5.2) 7



and 1.5 µL of 10 mM tris (2-carboxyethyl) phosphine (TCEP) for 1 h before use to reduce disulfide bonds. Then, a volume of 1.0 mL of the rGO-PdAu-GOx nanomaterials were added into P2 to react 16 h under magnetic stirring. Eventually, 500 µL of the prepared P2-rGO-PdAu-GOx complex was added into NaCl (250 µL, 100 mM) and Tris-HCl buffer solution (250 µL, 10 mM, pH 8.0) and stored at room temperature for 1 day. Most of the free P2 was removed after centrifugation and redispersion. The resulting solution was stored at 4 °C for further use. 2.3. Fabrication of the Dual Mode Integrated Lab-on-Paper Device. The ECL detection zone with excellent conductivity and enlarged surface area was fabricated by growing of Au NPs layer on the surfaces of cellulose fibers with HAuCl4 as metal sources and L-AA solution as reductant (the preparation conditions were optimized as shown in Figure S4). Concretely, the Au NPs seeds were obtained by using NaBH4 as reductant and sodium citrate as stabilizer according to previously reported literature.44 Then, 20 µL of the achieved Au NPs seeds solution was injected into the surface of the bare ECL detection zone, keeping it at ambient temperature for 60 min. Subsequently, 15 µL of freshly prepared growth solution containing HAuCl4 (12.2 mM) and L-AA (0.1 M) was dropped to the Au NPs seeded ECL detection zone with incubation at room temperature for another 60 min. After thoroughly rinsing with ultrapure water and dried at amnient temperature for 20 min, the Au NPs modified ECL detection zone was achieved and applied for the next experiment. The detailed design and fabrication procedure of the versatile lab-on-paper device is presented in the Supporting Information. Prior to the functionalization of ECL detection zone, an aqueous solution containing P1 (1 µM, 30 µL), Tris-HCl buffer (10 mM, pH 8.0, 500 µL) and TCEP (1 mM, 500 µL) were prepared. Afterwards, 20 µL of P1 solution was dropped onto the freshly prepared Au NPs modified ECL detection zone and incubated for 12 h at ambient temperature. During this process, the thiol-modified P1 was immobilized on Au NPs via Au-S bond. Then, the electrode was blocked with MCH (1 mM, 20 µL) for 2 h followed by washing with Tris-acetate buffer (10 mM, pH 8.0) to remove extra 8


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P1 and to obtain a well aligned DNA monolayer. Following that, the P1-AuNPs modified electrode was incubated with 20 µL P2-rGO-PdAu-GOx complex for 2 h at 37°C. Ultimately, the electrode was immersed in Tris-HCl buffer (10 mM, pH 8.0) for 5 min to reduce the nonspecific adsorption of P2-rGO-PdAu-GOx. For semiquantitation of colorimetric products, first of all, 20 µL TMB (20 mM) and 10 µL H2O2 (5 mM) were added to the visualization bar. The concentrations ranging from 0.5 nM to 2000 nM of Pb2+ solution were prepared by serial dilution. Afterwards, 20 µL of Tris-HCl solution (10 mM, pH 8.0) containing Pb2+ (10 nM) was cast onto the ECL detection zone and then incubated at 37 °C for 40 min to cleave the P2 strand. Following that, 30 µL 10 mM Tris-HCl buffer (pH 8.0) was added into the paper ECL detection zone and incubated for approximately 30 min at 30 °C, enabled the chromogenic reaction (Scheme 2D). The blank auxiliary tabs were further used to avoid the interference from external natural light and normalize lighting conditions to give more precise results accordingly. The color change was recorded instantly and the images were captured using camera. Furthermore, different concentrations of Pb2+ in tap water and river water were detected under optimum conditions. In order to achieve accurate quantification of Pb2+, the ECL detection zone was washed with ultrapure water thoroughly and then folded the reference tab down below the detection tab successively. Thereafter, Tris-HCl solution (pH 8.0, 40 µL) containing luminol and glucose was placed onto the paper ECL zone, followed by the respective ECL measurements. Ultimately, the ECL intensity was obtained by a MPI-B ECL analyzer with working potential from 0 to 0.6 V, and the voltage of the photomultiplier tube at 600 V (Scheme 2C). 3. RESULTS AND DISCUSSION 3.1. Working Principle of the Dual Mode Sensing. The dual mode lab-on-paper device is assembled by folding the specific tabs, as indicated by the green arrows in Scheme 1, to create the versatile origami-based paper device. The assay begins by injecting the pre-formed complex into the specific ECL detection zone and 9



visualization bar. Concretely, the thiol modified P1 was self-assembled onto the surface of the flower-like Au NPs modified ECL detection zone to hybridize with the rGO-PdAu-GOx labeled oligonucleotide forming the DNA double helix structure. Once Pb2+ was introduced into the platform, P2 would be cleaved. In the following, the channel tab was folded and covered the detection tab. The cleavable probe can transfer outward to the visualization bar and catalyze the decomposition of H2O2 to generate ·OH radicals along with the gray intensity enhancement of TMB, which permit prediction of Pb2+ concentration with naked eye. The screen-printed carbon working electrode, Ag/AgCl reference electrode and carbon counter electrode would be connected once the device fold at the predefined fold line and fill with solution. Then, GOx on uncleaved signal probe can catalyze the decomposition of coreactant H2O2 to produce abundant ·OH, an essential intermediate for luminol ECL reaction is proportional to the ECL intensity. The more P2, the deeper color of the TMB oxidation product and the weaker ECL signal would be obtained. 3.2. Morphology and Structure Characterization. The morphology and dimension of bare paper and Au NPs modified ECL detection zone were characterized by scanning electron microscopy (SEM), as shown in Figure 1A-D. Figure 1A reveals the porous cellulose fibers of bare paper, which afforded the particular attachment microenvironment for the growth of Au NPs. As can be found in Figure 1B-D, a dense layer of Au NPs, which are uniformly distributed on the paper fiber with flower like structures, were obtained on the paper fiber with an average dimension of about 500 ± 50 nm. Such unique architecture not only accelerates the electrons transfer but also enhances the surface area of paper detection zone to offer more active surface sites for specific DNAzyme. Energy dispersive X-ray spectroscopy (EDS) analysis of the obtained product shows that this material contained gold, carbon, and oxygen atoms, demonstrating the successfully preparation of Au NPs electrode (Figure 1E). The X-ray diffraction (XRD) was also characterized to further prove the structure of Au NPs. As described in Figure 1F, compared with bare paper (curve a), the characteristic diffraction peaks of Au NPs (curve b) appear at 38.4, 45.1, 64.8°, which are in good 10


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agreement with the (111), (200), and (220) crystallographic planes of cubic respectively (JCPDS card No 004-0784), indicating the successful construction of Au NPs working electrode. Furthermore, the comparison of the cross sectional image analysis of bare paper (Figure S3A) and Au NPs working electrode (Figure S3B to E) was carried out, as illustrated in Figure S3A-E. Obviously, flower like Au NPs penetrated the whole thickness of the paper with a continuous layer structure formed, furthermore verifying the fact that Au NPs grew on the paper successfully. Figure 2A&B reveal the TEM images of as-prepared Pd nanocrystals and PdAu bimetallic nanocrystals. It is clear that Pd nanocrystals exhibit typical nanocube structure with a mean edge length of 15-20 nm. Notably, the surface of the Pd nanocube is very smooth and free of bumps. As shown in Figure 2B, PdAu bimetallic nanocrystals are well defined as hexahedral nanostructures with narrow size distribution ranging from 20 nm to 25 nm. The crystal structure of PdAu bimetallic nanocrystals was characterized by XRD. Figure 2C shows that four reflections appeared at 39.3, 45.6, 66.4, and 78.8°, which were exactly located between Au (reference code 00-004-0784) and Pd (reference code 00-005-0681), implying the successful synthesis of PdAu bimetallic nanocrystals. Figure 2E and Figure S7 indicate the SEM images of as-prepared rGO and rGO-PdAu nanocomposites at different magnifications, from which obvious difference can be observed. Figure S7A&B show a typical neuronal-like shape of rGO with a smooth surface, while the surface becomes rough after hybridizing with PdAu bimetallic nanocrystals (Figure S7C&D). Figure 2F&G display the TEM images of rGO nanoplate and rGO-PdAu nanocomposites at different magnifications. It is obvious that rGO (inset of Figure 2F) has a well-defined plate-like morphology with ripples and corrugations, revealing the ultrathin nature of the rGO nanoplates. It can be clearly seen that PdAu bimetallic nanocrystals are highly monodispersed after assembled on the ultrathin rGO nanoplates. Moreover, compared with the EDS data of PdAu bimetallic nanocrystals (Figure 2D), rGO-PdAu-GOx nanomaterials shows the C and O element peaks apart from Au and Pd element peaks (Figure 2H). The cross sectional images and EDS data of ECL detection zone after incubated with P1 and P2-rGO-PdAu-GOx-Pb2+ are 11



illustrated in Figure S3F-I (SEM images are illustrated in Figure S8). Such phenomenons all demonstrate that the nanomaterials have been synthesized successfully. 3.3. Comparison of the ECL Response with Different Signal Amplification Strategies. Signal amplication plays an important role to achieve high sensitivity and low detection limit of the integrated sensing device. In the assay, rGO-PdAu nanocomposites were employed as effective nanocarriers to load GOx and P2. To compare and demonstrate the superiority of the proposed rGO-PdAu nanocomposites, contrast experiments were conducted by comparing the ECL intensity of the lab-on-paper device incubated with five different labeled probes in the same working buffer. As shown in the Figure 3A, the ECL values of the device incubated with P2-rGO-PdAu-GOx probe is 4800.0 au (curve e), while that decreases to 1197.33, 1438.34, 1972.89 and 3188.76 au, when incubated with P2-Au NPs-GOx (curve a), P2-Pd NPs-GOx (curve b) and P2-PdAu-GOx nanocomposites (curve c) and P2-rGO-AuNPs-GOx (curve d), respectively. That proves that the catalytic ability of PdAu bimetallic nanocrystals were more excellent than Au NPs and Pd NPs, while rGO plays crucial role for the catalytic amplification of ECL intensity. By the effective integration of rGO and PdAu bimetallic nanocrystals, the proposed P2-rGO-PdAu-GOx probe showed the most significant electrocatalytic efficiency. All the phenomenons can be attributed to the following reasons: (i) rGO nanoplates as the loading carrier of nanomaterials can prevent the aggregation of noble metal NPs, such as Au NPs, Pd NPs, and PdAu bimetallic nanocrystals effectively (Figure 2F). (ii) rGO nanoplates possess good conductivity and large area. Under the synergetic catalysis between rGO and well-dispersed PdAu bimetallic nanocrystals, more enhanced catalytic performances toward H2O2 could be realized. Meanwhile, more GOx could be loaded to catalyze the oxidation of glucose to produce H2O2 as coreactant for luminol, implementing high sensitivity of the dual mode lab-on-paper device. Figure 3B shows the ECL intensity obtained from the integrated lab-on-paper device in the presence and absence of target Pb2+. A significant ECL signal appeared 12


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(curve a) in the absence of Pb2+. However, with addition of Pb2+, a decreased ECL signal was obtained (curve b), indicating the successful immobilization of GOx on P2-rGO-PdAu probe as well as the feasible construction of the paper device for Pb2+ determination. As illustrated in Figure 3C, the typical CVs of the aforementioned electrodes in Tris-HCl buffer at a scan rate of 100 mV s-1 were compared. As judged from the measured current, there was no substance with electrochemical activity in the electrodes and solution (P2-rGO-PdAu (curve a), P2-rGO-PdAu-MCH (curve b)). However, a pair of well-defined redox peaks (curve c) could be observed in the working potential range while the probe of P2-rGO-PdAu-GOx was introduced into the electrode. Which indicated that the ECL signal was generated relying on the oxidation of glucose by GOx. CV and EIS characterization for each immobilized stages are shown in Figure S11. 3.4. Analytical Performance of the Integrated Lab-on-Paper Device. To quantitatively investigate the peroxidase-like activity of enzyme mimics, the steady-state kinetic parameters of the PdAu bimetallic nanocrystals was studied and the initial reaction rates versus the H2O2 concentrations were presented in Figure 4 A&B. For comparison, Km values of other enzyme mimics and HRP reported previously were listed in Table S1. The Km value of the PdAu bimetallic nanocrystals for H2O2 was lower than other NPs-based peroxidase mimetics and natural enzyme apparently, implying the fact that PdAu bimetallic nanocrystals possess more preferable peroxidase-like activity. In order to confirm the enhancement catalytic oxidation of PdAu nanocrystals on TMB, Pb2+ concentration-dependent gray intensity was examined to implement visual prediction before ECL precise detection (Figure S5). On the basis of the peroxidase-like activity of PdAu bimetallic nanocrystals, the Pb2+ concentration-dependent gray intensity was investigated to implement visual prediction. As displayed in Figure 4C, the gray intensity increased gradually with the increasing of Pb2+ concentration because the amount of cleaved signal probe was proportional to Pb2+ concentration. The gray intensity degree linearly responds to the logarithmic value of Pb2+ concentration ranging from 5 to 2000 nM with a correlation 13



coefficient of 0.9976. The regression equation was Y= 71.75+7.62 lgc. As presented in the inset of Figure 4C, it is easy to realize visual prediction in the all-in-one lab-on-paper device without any complicated instrument (comparison diagrams of the colorimetric color variance with or without shading tab are shown in Figure S9&10). As plotted in Figure 4D, the rGO-PdAu nanocomposits in this method was observed to be very stable and revealed high catalytic activity even after incubation with pH ranging from 3.0 to 10 and temperature increased from 4 to 80 °C, indicating the robust nature of the rGO-PdAu nanocomposits suitable for a broad range of applications. Thereafter, in order to achieve the maximal detection performance of Pb2+, a series of conditional optimizations including the pH value of reaction solution, reaction temperature, incubation time, and the volume of P2-rGO-PdAu-GOx complex were carried out as indicated in Figure S6. Under the optimal conditions, the developed lab-on-paper device retained an excellent analytical performance for Pb2+ determination with the linear range of 0.5 to 2000 nM (Figure 5A). The linear equation was I = 1599.95 +1511.55 lgc with a correlation coefficient of 0.9977, where I was the changes of ECL intensity and c was the concentration of Pb2+. The estimated limit of detection (LOD) reached as low as 0.14 nM, which was estimated using 3σs/S criteria (σs is the standard deviation of intensity in a blank solution and S is the slope of the linear calibration curve, n = 11). In addition, the operational stability as one of the vital factor of the ECL lab-on-paper device was evaluated in this work. Figure 5C shows that ECL intensities obtained from seven different concentrations of Pb2+ solution have a relatively stable signal, suggesting that the prepared integrated lab-on-paper device possess an acceptable stability for Pb2+ concentration measurement. The relative standard deviations (RSD) ranged from 1.2 to 3.1 as the concentration increases, indicating an excellent stability of the device. Additionally, the reproducibility of the sensing system was estimated. Seven lab-on-paper devices with 10 nM Pb2+ prepared in the same batch were examined under the same experimental conditions. A semblable ECL signal was received with the RSD of 2.9%, illustrating the outstanding reproducibility 14


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of the integrated lab-on-paper device. Meanwhile, the interference experiments were carried out to evaluate the specificity of this dual mode integrated lab-on-paper device, in which different concentrations of other metal ions (K+, Mn2+, Zn2+, Cu2+, Ca2+, Ni2+, Cr2+ and Cd2+) were opposing to the reaction mixture containing Pb2+ (10 nM) under the optimal experimental conditions. As described in Figure 5D, interfering ions have negligible effect on the ECL signal. The above consequence illustrated that the lab-on-paper device possesses high tolerant concentrations of other interfering ions, suggesting the detection system exhibits remarkably high selectivity toward Pb2+. 3.5. Application of the Dual Mode Lab-on-Paper Device in Real Samples. To further evaluate the feasibility and capability of proposed method in actual samples, the constructed dual mode lab-on-paper device was employed for detecting Pb2+ in tap water and river water samples. Prior to use, the river water samples were centrifuged for 15 min at 12,000 rpm. After that, Pb2+ in a complicated sample matrix was determined by dispensing different concentrations of target Pb2+ with the prepared samples via a standard addition method. The ECL intensities were parallelly investigated in 10 mM Tris-HCl buffer (pH 8.0) containing glucose and luminol. As shown in Table 1, the recoveries (defined as the ratio between the found concentration and the added concentration) are calculated to be in the range of 96.74 to 97.62 with the RSD values from 3.05 to 5.31 for tap water, ranging from 96.13 to 99.52 with the RSD values from 2.17 to 5.28 for river water, suggesting the good reliability and accuracy of the proposed strategy for Pb2+ detection in real complicated samples. CONCLUSION In summary, a flexible dual mode lab-on-paper device was constructed with the aid of recognition between Pb2+ and specific DNAzyme. Not only the channel tab but also the reference tab can be folded into the desired pattern, resulting in dual mode versatile device. Benefitting from prominent features of the as-prepared nanomaterials, robust yet highly efficient signal amplification for Pb2+ detection can be implemented. It is believed that such approach for integrating dual detecting techniques through 15



printing on paper enables the construction of various fantastic paper devices and capabilities for environmental engineering assay. With reliance on the instinctive properties proposed above, next, portable, and sufficient equipment can be integrated for on-chip functions, which will be expanded to explore an extensive range of metal ion targets through the judiciously engineered ECL lab-on-paper technique. ASSOCIATED CONTENT Supporting Information Fabrication of the integrated lab-on-paper device, optimization of the detection conditions; CV and EIS characterization, comparison of the Km of various enzyme mimics; (Figures S1-S11, Tables S1&S2) AUTHOR CONTRIBUTION Corresponding Author *Tel: +86-531-82767161; Fax: +86-531-82765956. *E-mail address: [email protected] ORCID Jinghua Yu: 0000-0001-5043-0322 Author Contribution #

J.X. and Y.Z. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors are thankful for support from National Natural Science Foundation of China (51502112, 21775055), the program for Taishan Scholars, and Key Research and Development Program of Shandong Province, China (2016GGX102035).

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Table Table 1. Detection of Pb2+ in real samples with the lab-on-paper device. Samples

Added / nM

Found/ nM

Recovery/% RSD/%

tap water 1

1.0

0.9674

96.74

3.73

tap water 2

5.0

4.852

97.04

3.05

tap water 3

10

9.762

97.62

5.31

river water 1

1.0

0.9813

98.13

2.17

river water 2

5.0

4.976

99.52

3.41

river water 3

10

9.613

96.13

5.28

22


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Figure captions Scheme 1. Schematic Representation of Fabrication Procedures of the Lab-on-Paper Device and Dual Mode Sensing Mechanism.

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Scheme 2. (A) Schematic Layout, Size, and Shape of the Integrated Lab-on-Paper Device. (B-D) Photographs of the Prepared Integrated Lab-on-Paper Device with Constructed Three Electrodes System. Where the Working Electrode Was Obtained by in-situ Growth Method, Reference and Counter Electrodes Were Fabricated by Means of Screen-Printing Technique.

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Figure 1. Representative SEM images of (A) bare paper surface, (B-D) flower-like Au NPs modified paper surface. (E) EDS of Au NPs modified paper surface. (F) XRD analysis on bare paper (curve a) and Au NPs modified paper surface (curve b).

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Figure 2. Representative TEM images of (A) Pd nanocubes, (B) PdAu bimetallic nanocrystals. (C) XRD analysis of PdAu bimetallic nanocrystals. (D) EDS of PdAu bimetallic nanocrystals. (E) SEM image of rGO-PdAu nanocomposites. (F, G) TEM images of rGO-PdAu nanocomposites under different magnifications, inset: rGO nanoplate. (H) EDS of rGO-PdAu nanocomposites.

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Figure 3. (A) ECL comparison of the lab-on-paper device incubated with different labeled probes: (a) P2-AuNPs-GOx (b) P2-PdNPs-GOx, (c) P2-PdAu-GOx, (d) P2-rGO-AuNPs-GOx (e) P2-rGO-PdAu-GOx . (B) ECL characterization in Tris-HCl buffer (0.1 M, pH 8.0): (a) without target Pb2+ (b) with target Pb2+ (10 nM). (C) CVs characterization in Tris-HCl buffer (10 mM, pH 8.0): (a) P2-rGO-PdAu, (b) P2-rGO-PdAu-MCH, (c) P2-rGO-PdAu-GOx-MCH.

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Figure 4. (A) Steady-state kinetic assay of PdAu bimetallic nanocrystals. The concentration of TMB was 20 mM and the concentration of H2O2 was varied. (B) Double reciprocal plots of activity of PdAu bimetallic nanocrystals with the concentration of one substrate fixed and the other varied. (C) Calibration curve for colorimetric Pb2+ detection. Inset: color imagings of the lab-on-paper device incubated with different concentrations of Pb2+, from a to g: 5, 10, 50, 100, 500, 1000, 2000 nM, respectively. (D) Intensity stability of the prepared PdAu bimetallic nanocrystals under pH values from 3.0 to 10 for 5.0 h and temperature from 4 to 80 °C for 5 h.

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Figure 5. (A) ECL profiles of the lab-on-paper device incubating Pb2+ with different concentrations of (a-i): 0, 0.5, 5, 10, 50, 100, 500, 1000 and 2000 nM. (B) The corresponding calibration plot for Pb2+ detection. All ECL signals were detected in Tris-HCl (10 mM, pH 8.0) buffer. (C) The ECL stability of the lab-on-paper device to various concentrations of Pb2+ under consecutive cyclic potential. (D) Selectivity of the lab-on-paper device toward different interfering substances.

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Graphic Abstract


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Colorimetric and Electrochemiluminecence Dual Mode Sensing of

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