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Sensors and Actuators B 237 (2016) 308–317

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo u r nal homep age: www.elsev ier .com/ locate /snb

aper-based bipolar electrode-electrochemiluminescence (BPE-ECL)evice with battery energy supply and smartphone read-out: Aandheld ECL system for biochemical analysis at the point-of-care

evel

u Chen, Chunsun Zhang ∗, Da Xing ∗

OE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

r t i c l e i n f o

rticle history:eceived 25 March 2016eceived in revised form 15 June 2016ccepted 18 June 2016vailable online 19 June 2016

eywords:ipolar electrodelectrochemiluminescenceaper microfluidics

a b s t r a c t

In this work, a handheld paper-based bipolar electrode-electrochemiluminescence (P-BPE-ECL) systemhas been proposed. In this system, a rechargeable battery is used for power supply, while a smartphone isapplied for read-out of ECL signal. For the P-BPE-ECL, the carbon ink-based BPE and driving electrodes arescreen-printed on the paper, and the wax-screen-printing is employed to fabricate microfluidic channelson electrode-patterned paper. Moreover, the luminol/H2O2-based ECL reaction is applied to demonstratethe quantitative ability of the P-BPE-ECL system. Under optimized conditions, H2O2 can be determinedover the range of 5–5000 �M, with a detection limit of 1.75 �M, and relative standard deviations (RSDs)of 7.59%, 3.82% and 4.93% for 10, 100 and 1000 �M H2O2 (n = 5). Thus, this system has an acceptablesensitivity, dynamic range, stability and reproducibility. Finally, the applicability of the P-BPE-ECL system

echargeable-battery energy supplymartphone-based read-outetection of H2O2 or glucose

is demonstrated for detection of glucose in phosphate buffer solution (PBS) and artificial urine (AU)samples, with the detection limits of 0.017 mM and 0.030 mM, respectively. Moreover, the P-BPE-ECLsystem has the ability to perform high-throughput detection of glucose. Therefore, the developed systemcan provide a new platform for many applications such as point-of-care testing, health diagnosis andenvironmental monitoring.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

Electrochemiluminescence (ECL) is a technique resulting fromhe combination of electrochemistry and chemiluminescence1–3]. It has several attractive features such as absence of back-round signal, better control over the position or time of lightmission, simultaneous acquisition of current signal and light sig-al, compatibility with solution-phase and thin-film formats, andpportunities to enhance intensity with nanomaterials [3,4]. And,hese features make ECL a highly sensitive and selective analytical

ethod for quite a wide range of applications in the areas of clini-al diagnostics, food and water testing, environmental monitoring,

iowarfare agent detection, scientific research and so on.

Currently, the vast majority of ECL assays are performed inhree-electrode or two-electrode electrochemical (EC) systems

∗ Corresponding authors.E-mail addresses: zhangcs@scnu.edu.cn, zhangcs scnu@126.com (C. Zhang),

ingda@scnu.edu.cn (D. Xing).

ttp://dx.doi.org/10.1016/j.snb.2016.06.105925-4005/© 2016 Elsevier B.V. All rights reserved.

[1–3]. However, a relatively small number of ECL assays areachieved by using bipolar electrode (BPE)-based ECL (BPE-ECL) sys-tems that can be performed in either an open [5–20] or a closed[21–25] cell. In the former BPE-ECL systems, the BPE is placed insidea microchannel. By contrast, when using a closed bipolar cell, twopoles of a BPE are located in two separate compartments. In com-parison with conventional ECL that pays attention to the redoxreactions at the working electrode, the BPE-ECL technique focuseson the coupling of EC and ECL reactions occurring at two oppositepoles of a BPE. Thus, a BPE-ECL sensing platform usually possessesseveral inherent advantages such as relatively low cost, no needfor direct contact of external potential instruments with sensingelectrodes, ease of integration to portable devices, and possibilityof high-throughput analysis.

For BPE-ECL, BPEs are usually set up in microfluidic devices.The first BPE-ECL microfluidic sensor was developed by Manz andco-workers by using a “U”-shaped platinum film BPE across the

open cell of glass chip [5]. Since then, a series of BPE-ECL microflu-idic devices have been developed by several groups. For example,microfluidic chips used in the Crooks, Wang, and Fang groups

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ere comprised of a poly(dimethylsiloxane) (PDMS) block bondedrreversibly to a glass slide, in which standard lithographic and soft-ithographic approaches were applied to prepare the indium tinxide (ITO) [6–9,23,25] or gold [10–13,21,24] BPEs onto the glassubstrate, and fabricate the PDMS channels, respectively. In the Xuroup, the PDMS [14,16] or glass [15,18] molds with positive surfaceeliefs have been used to fabricate the PDMS channel, and patternhe ITO BPE onto the glass. For BPE-ECL microfluidic devices fromther groups [17,19,20], the BPE could be assembled into a capillaryube or certain cell to perform BPE-ECL.

Within the abovementioned BPE-ECL devices, new functionalityan be introduced to BPE-ECL detection by changing either the ECell or the shape of the BPE. However, these devices often requirehe bonding/sealing of the channel to form an enclosed structure,he implementation of micro-junction at the channel ends, and anxternal fluidic driving unit to deliver a solution into the channel.hus, complicated chip fabrication processes and high-cost elec-rode materials are often required. Moreover, expensive peripheralquipments (including ECL analyzer, CCD camera or photomulti-lier tube (PMT) for data collection, syringe, vacuum or peristalticumps for the injection or extraction of solution, and as well bulkyC power source and even EC workstation for the potential controletween the driving electrodes) are usually required. Therefore, it

s necessary to develop special power sources, signal acquisitionodes, and chip devices for user-friendly BPE-ECL measurement.Since the first paper-based ECL microfluidics was introduced by

hen and co-workers [26], paper-based ECL devices have attractedncreasing attention and interest [27–40]. However, such devicesre still in their early stage of development. Thus, there are stillressing needs for new ECL method for paper devices. As mentionedbove, BPE-ECL has become as a new type of ECL detection methodn recent years, and has shown great prospects for potential applica-ions [41,42]. In general, the basic components of a BPE-ECL systemnclude an electrical energy supply, a bipolar EC chamber and anptical detector. In addition, various types of BPE-ECL chambersan be used for different BPE-ECL studies.

In this work, a handheld paper-based microfluidic BPE-ECL (P-PE-ECL) device, which is integrated with a rechargeable battery

or power supply and a smartphone for the read-out of ECL signal,as been explored. This P-BPE-ECL system has several attractive

eatures as follows: (i) the paper channel is not closed, thus provid-ng an easy-to-operate sampling, and with no need for an externalump; (ii) the paper-based screen-printing fabrication is simple,apid, and cheap, without any complicated fabrication processes;iii) the P-BPE-ECL device needs a low driving voltage and smallnergy-consumption, and thus the fully-charged battery can runor tens of hours; and (iv) the ECL signal is collected by an inex-ensive smartphone, and can be wirelessly transmitted to theersonal computer (PC). In addition, the general features of paperevices (such as low-cost, disposability, portability, biodegradabil-

ty) [43–45] also belong to the P-BPE-ECL devices. Together withhese features, the P-BPE-ECL system is suitable for portable bio-hemical analysis at the point-of-care (POC) level. In the presentedase, the proposed ECL system has been successfully applied foretermination of H2O2, with acceptable sensitivity, dynamic range,tability and reproducibility. In addition, the applicability of P-BPE-CL is further demonstrated for detection of glucose in differentamples, and for the potential of high-throughput glucose screen-ng. As we know, the determination of H2O2 or glucose is of greatmportance in many fields, and has attracted increasing attention.nd, the reaction mechanism for the luminol/H2O2-based ECL sys-

em was proposed as follows [46]:

uminol−e−−H+ → Diazoquinone (1)

2O2 + Diazoquinone → Luminolendoperoxide (2)

ors B 237 (2016) 308–317 309

Luminolendoperoxide → N2 + 3-aminophthalate∗ (3)

3-aminophthalate∗ → 3-aminophthalate + h� (4)

2. Experimental section

2.1. Materials and chemicals

Conductive carbon ink (CNB-7, <60 �/sq) was purchased fromXuzhou Bohui New Materials Technology Co., Ltd. (Xuzhou, China).Whatman chromatography paper #1 (20 cm × 20 cm) was obtainedfrom GE Healthcare Worldwide (Shanghai, China). Wax crayons(Detong Co., Ltd., Guangzhou, China) and smooth utensil werebought from a local stationery store. Sodium hydroxide and sodiumcarbonate anhydrous were produced by Guangzhou ChemicalReagent Factory (Guangzhou, China). Potassium chloride and �-d-Glucose (anhydrous, 96%) were purchased from Sigma-Aldrich(St. Louis, MO, USA). Luminol (97%), sodium bicarbonate (> 99%),phosphate buffer solution (PBS) (20×), H2O2 (> 30%, v/v), glucoseoxidase (GOD) (CAS# 9001-37-0, 50 KU), sodium dihydrogen phos-phate monohydrate, magnesium sulfate heptahydrate, sodiumhydrogen phosphate, sodium sulfate anhydrous, sodium oxalate,calcium chloride dihydrate, ammonium chloride, sodium chlorideand sodium citrate dehydrate were purchased from Sangon BiotechCo. Ltd. (Shanghai, China). Urea (99%), uric acid (98%) and creatinine(99%) were from J&K Chemical Ltd. (Beijing, China). All chemicalswere of analytical grade and used as received without further pre-treatment.

2.2. Instrumentation

The YH-946B microcomputer temperature-controlled heatingboard was produced by Guangzhou Yihua Electronic EquipmentsCo., Ltd. (Guangzhou, China). The DHG-9035A oven was purchasedfrom Shanghai Tensuc Experimental Instrument ManufacturingCo., Ltd. (Shanghai, China). The PB-10 Sartorius Basic pH meter wasobtained from Sartorius Scientific Instruments (Beijing) Co., Ltd.(Beijing, China). The lithium battery (XSL-12.6V, 4.4 ah) was fromXinshengli Power Supply Co., Ltd. (Shenzhen, China). The MI 2SCsmartphone was produced by Beijing Xiaomi Science and Technol-ogy Co., Ltd. (Beijing, China). The electronic circuit model (YS-05)for voltage control and display was obtained from YusongchuangdaElectronics Co., Ltd. (Shenzhen, China). The screens for screen-printing the paper devices were custom-made from LianchangPrinting Equipment Shop (Guangzhou, China). A water-purificationsystem (≥18 M�, ELGA PURELAB® Option-R15, London, UK) wasused to prepare deionized (DI) water. A scanning electronic micro-scope (SEM) (ZEISS Ultra 55, Carl Zeiss, Germany) was used formicroscopic analysis of paper devices.

2.3. Design and assembly of the handheld P-BPE-ECL system

The handheld P-BPE-ECL system is illustrated in Fig. 1A, whichmeasures 20 cm × 15 cm × 7 cm, and weighs about 0.5 kg. This sys-tem is mainly composed of five subsystems: a rechargeable lithiumbattery, an electronic circuit model, a smartphone, a paper device,and an instrument container. The subsystems are described indetails as follows.

The rechargeable lithium battery (size:6.5 cm × 5.4 cm × 3.7 cm; weight: 0.26 kg) (Fig. 1B) is used asthe power supply of the P-BPE-ECL reaction, and can last about146 h for the present applications in which the operating current

is 30 mA. Therefore, such a battery may be highly suitable whenit operates for the POC field. To maintain, adjust and display thedriving voltage required for P-BPE-ECL, an electronic circuit model(Fig. 1B) for voltage control is applied, including the xl4015 chip,

310 L. Chen et al. / Sensors and Actuators B 237 (2016) 308–317

Fig. 1. Scheme of the proposed P-BPE-ECL system. (A) Diagram of the handheld system: (1) smartphone; (2) instrument container; (3) charging cable; (4) rechargeablel -ECL p( controo conta

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ithium battery; (5) electronic circuit model for voltage control and display; (6) BPEB) Photo of the battery and electronic circuit model: (1) potentiometer for voltage

utput voltage. (C) Diagram of the electronic circuit. (D) Picture of the paper device

olar capacitor, non-polar capacitor, inductance coil, zener diode,otentiometer, digital tube and running lights. The typical applica-ion circuit of the electronic model is shown in Fig. 1C. Here, the R2otentiometer can be adjusted to obtain a certain output voltageor the P-BPE-ECL. In the case of an adequate driving voltage, the-BPE-ECL signal can be collected by the smartphone that is fixedn the cover of instrument container. And, the complementaryetal-oxide semiconductor (CMOS) camera of the smartphone is

laced right above the BPE (Fig. 1D), and a distance of 30 mm apartrom each other is required to ensure that the acquired image islear. Benefiting from the unique design of the system and theide field-of-view characteristic of the smartphone, the P-BPE-ECL

mage can be well captured, without any mechanical movementf the paper device or the imaging system. Moreover, the capturedmages can be wirelessly transmitted to the PC that can rapidlyrocess these images to yield the desired analytical curves. Ithould be noted that except the smartphone, the components ofhe handheld ECL system cost less than $20.

.4. Principle for the P-BPE-ECL

Fig. 2 depicts the principle for the P-BPE-ECL sensing of H2O2hose detection is often the basis of other target biosensing. For

his sensing, the BPE, as an electronic conductor without anyirect electrical connection, is located inside a microchannel whoseimensions are small enough to ensure a high resistance to ionic

urrent flow. Because the channel resistance is high, most of Etot

the voltage between the driving electrodes) is dropped linearlylong the channel length (Lchannel). Thus, the potential difference�Eelec) across the length of BPE (Lelec) can be simply estimated

aper device; (7) device support; and (8) wireless transmission of ECL signal to PC;l; (2) digital-tube for voltage display; (3) positive output voltage; and (4) negativeining two sets of P-BPE-ECL units.

as �Eelec = Etot (Lelec/Lchannel). If the value of �Eelec is sufficientlyhigh, a pair of redox processes occur simultaneously at the poles ofthe BPE. In our case, after the reagents are injected into the paperchannel, a driving voltage can activate simultaneous oxidation ofthe lumimol/H2O2 and reduction of the reducing agent (such asO2) at the anodic and cathodic poles. And, the BPE-ECL signal at theanodic pole is captured by the smartphone for visual detection.

2.5. Preparation of paper devices

The screen-printing fabrication process for BPE-ECL paperdevices is shown in Fig. 3. Carbon ink-screen-printing was usedto fabricate the electrodes onto the paper, while wax was uti-lized as the paper hydrophobization to construct the hydrophobicbarrier on the paper. The shapes of electrode and hydrophobicbarrier on the paper were designed by Adobe illustrator CS5 andthen were applied to produce the screens (300 mesh of polyesteron aluminium frame). As shown in Fig. 3, the paper was cut intoa suitable size (100 mm × 100 mm), and the electrode array wasscreen-printed on the paper through the electrode-screen, followedby curing this array in a 90 ◦C oven for 10 min and cooling it to roomtemperature (Steps 1–5 in Fig. 3). Subsequently, the as-preparedelectrode-patterned paper was placed below the channel-screen,and solid wax was rubbed through the screen onto the paper (Steps6, 7 in Fig. 3). To allow more waxes to print onto the paper, a smoothutensil was used to further press and rub the screen. Next, the wax-

screen-printed paper was placed onto the heating board togetherwith the screen, and heated at 90 ◦C for about 3 s to melt the waxinto the paper (Steps 8, 9 in Fig. 3). Note that the wax patterns couldbe fabricated on the electrode-containing paper in small bulk.

L. Chen et al. / Sensors and Actuat

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ig. 2. Schematic description of the design and principle of the P-BPE-ECL for bio-hemical analysis.

.6. P-BPE-ECL assay procedure

After the completion of device fabrication, the P-BPE-ECL assayould be performed. A detailed procedure is described below. The

ig. 3. Schematic illustration of the screen-printing fabrication for the P-BPE-ECL devices. (nto the paper; (3) the carbon ink-printed paper was separated from the electrode-screenoom temperature to form the electrode-patterned paper; (6) the channel-screen was pland then a smooth utensil was used to further press and rub the screen; (8) the paper wasere taken away from the heating board, and separated from each other to the desired p

ors B 237 (2016) 308–317 311

as-prepared paper device was fixed onto a support within theinstrument container, and two driving electrodes were correspond-ingly connected to the positive/negative output voltage supplies ofthe electronic circuit model that had been connected to the bat-tery. Then, a 25-�L mixture of luminol (0.5-12.5 mM) and H2O2(0.005-5.0 mM), which was made in 0.1 M Na2CO3-NaHCO3 buffersolution, was dropped onto the hydrophilic channel. For glucosedetection, 3 �L GOD (1 U/�L) was immobilized on the BPE anode bythe physical adsorption. Here, 1 �L of GOD solution was spotted onthe surface of the anode, followed by about 2 min of drying at roomtemperature. Such process was repeated three times to accomplishthe GOD immobilization, followed by preserving the paper device at4 ◦C in a refrigerator prior to use. Then, 12.5 �L glucose (0.1–5 mM)that was made in PBS (pH 6.9) or AU (pH 6.0) was added to thechannel for production of H2O2. After a 30 s wait, 12.5 �L lumi-nol was added to the channel. Before the device was powered, a30–60 s wait was required to ensure that the whole channel waswell filled with the reaction solution. Next, an appropriate drivingvoltage was imposed to obtain the stable ECL at the BPE anodicpole, and the ECL signals were captured by the smartphone andwirelessly transmitted to the PC for analysis.

2.7. Data acquisition and analysis

The captured images were recorded in the JPEG format and ana-lyzed by a method similar to that reported in a previous work [21].Each desirable image was cut to be a single picture that had a sizeof 10.6 mm × 10.6 mm (length × width), and contained the wholelight-emitting zone on the BPE anode. The pretreated picture with

an area of about 1600 pixel was analyzed by the Image J softwarewhich converted the brightness of each pixel on the micrograph tophoton counts. The P-BPE-ECL intensity was calculated by integrat-ing the photon count for each pixel of the micrograph.

1) The electrode-screen was placed on the paper; (2) carbon ink was screen-printed; (4) the carbon ink-printed array was cured in an oven; (5) the array was cooled toced onto the electrode-patterned paper; (7) the screen was rubbed with a crayon,

placed on the heating board together with the screen; and (9) the screen and paperaper devices.

312 L. Chen et al. / Sensors and Actuators B 237 (2016) 308–317

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ig. 4. Picture (A) and SEM images (B–D) of a screen-printed P-BPE-ECL device. Panenk-screen-printed electrodes, respectively.

. Results and discussion

.1. Design, fabrication and characterization of P-BPE-ECL device

In general, the design and fabrication of BPE-ECL chamberss crucial to a BPE-ECL system. In our study, carbon ink-creen-printing was applied to fabricate the electrodes onto theaper, while wax-patterned BPE-ECL chambers were directlyax-screen-printed onto the electrode-containing paper. By two

creen-printing processes, our P-BPE-ECL devices could be well fab-icated. And, a typical screen-printed P-BPE-ECL device is shownn Fig. 4A. It has been shown that the presented fabrication

ethod could provide a good hydrophilic/hydrophobic contrasto define the paper device in which the total length of the chan-el was 22 mm, the width of the BPE-containing channel was

mm, the sizes (length × width) of BPE and driving electrodeere 3 mm × 1 mm and 4 mm × 5 mm, respectively. In addition,

fter the BPE and driving electrodes were one-step constructednto the paper, the wax barrier-based chamber could be pro-essed onto the paper to well contain one BPE and two drivinglectrodes. To investigate the microscopic characteristics of theevice, the SEM images of three different functional zones werebserved (Fig. 4B–D). Fig. 4B shows the porous structures andicrofibers of the pure cellulose paper, revealing that the bare

aper possessed high surface area with rough cellulose fibersnd could offer an excellent hydrophilic microemvironment. Itan be seen from Fig. 4C that owing to the porous structuref the paper, the screen-printed wax could well penetrate intohe paper to form the hydrophobic barrier. As shown in Fig. 4D,

continuous and dense carbon conducting layer on the sur-

ace of the paper was observed, indicating that a good coveragef carbon materials on the surfaces of the cellulose fibers wasbtained.

(D) indicate the structures of the pure paper, wax-screen-printed paper and carbon

3.2. P-BPE-ECL assay optimization

In the P-BPE-ECL system, the smartphone was applied for read-out of ECL signal, with no need for a high-cost and complex PMT orCCD detection system. Note that the smartphone as a commonplaceand portable detector has been effectively employed for colori-metric detection in paper devices, but it has fairly rarely used fordetection of paper-based ECL signals [26]. As we know, ECL is usu-ally performed in the dark. In our case, therefore, the smartphonewas flexibly fixed above the cover of instrument container, andits CMOS camera was aligned to the BPE anode. To clearly capturethe ECL emission, the distance between the CMOS camera and theanode should be suitable. It has been observed in Fig. S1 that at a dis-tance of less than 30 mm, the camera was unable to focus well andthus the captured photo became obscure. On the contrary, whenthe distance was greater than 30 mm, the field of view of the cam-era would exceed the area of the paper device, and consequentlyit was difficult to capture the stronger ECL signal, and moreover itwas possible to bring other interference signals. Therefore, 30 mmwas chosen as the optimal distance.

In the presented sensing platform, the driving voltage is one ofthe main factors that affect the P-BPE-ECL signals. Fig. 5A showsthe P-BPE-ECL responses for different driving voltages. It has beenfound that the ECL intensity was increased with increasing the driv-ing voltage from 3.0 V to 7.0 V, which may be due to the increasein the EC oxidation rate of luminol to form 3-aminophthalate*

[47–49]. When the driving voltage was lower than 3.0 V, no ECLsignal was obtained, possibly because the used voltage was notsufficient to initiate the EC oxidation of luminol. However, whenthe driving voltage was above 7.0 V, the ECL signal decreased. It hasbeen reported that a high driving voltage can initiate background

reactions, such as the oxidation of water, and this in turn resultsin the formation of oxygen that both chemically and physicallyinterferes with ECL emission [21].

L. Chen et al. / Sensors and Actuators B 237 (2016) 308–317 313

Fig. 5. Dependence of ECL images (i) and intensities (ii) on the driving voltage (A), pH of buffer solution (B), and concentration of luminol (C). In panel (A), Driving voltage:3 . In pa5 Drivinfi

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.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, and 9.0 V; pH: 9.0; [Luminol]: 5 mM; [H2O2]: 1 mM mM; [H2O2]: 1 mM. In panel (C), [Luminol]: 0, 0.5, 1, 2.5, 5, 7.5, 10, and 12.5 mM;ve independent experiments.

Besides the above mentioned factors, the concentrations ofome reagents also influence the P-BPE-ECL intensities. As shown inig. 5B, the pH of Na2CO3-NaHCO3 buffer solution that was used torepare the mixture of luminol and H2O2 was demonstrated to have

significant effect on the ECL intensity. The ECL intensity increasedith an increase in the pH value from 7.50 to 10.50. The reason

or this phenomenon is that luminol-based ECL reaction is morefficient in alkaline media [50]. However, a weaker ECL signal wasbserved when the pH was higher than 10.50, since the highly alka-ine surroundings would result in the decrease of the fluorescent

nel (B), pH: 7.5, 8.0, 9.0, 10.0, 10.5, 11.0 and 11.5; Driving voltage: 7.0 V; [Luminol]:g voltage: 7.0 V; pH: 10.5; [H2O2]: 1 mM. The error bar is the standard error from

quantum yield of the phthalate ion [51]. Therefore, pH 10.50 wasoptimized for the P-BPE-ECL. In the presented sensor, the degreeof ECL signal was also dependent on the concentration of luminol(Fig. 5C). It has been observed that the ECL intensity almost lin-early enhanced with increasing the luminol concentration from 0 to7.5 mM, which may be due to that the rate of luminolendoperoxide

formation increased with increasing luminol concentration [52].It should be noted that in the absence of luminol, the P-BPE-ECLonly exhibited a luminescence value close to the background sig-nal. When the luminol concentration was higher than 7.5 mM, the

314 L. Chen et al. / Sensors and Actuators B 237 (2016) 308–317

Fig. 6. ECL images (i) and intensities (ii) for detection of H2O2 (A), and glucose in PBS (B) and AU (C) analytes. In panel (A), [H2O2]: 0, 5, 10, 20, 50, 100, 200, 500, 1000, 2000,and 5000 �M; Driving voltage: 7.0 V; pH: 10.5; [Luminol]: 7.5 mM. In panels (B) and (C), the pH values were 11.0 and 11.25, respectively; [Glucose]: 0, 0.1, 0.5, 1, 2, and 5 mM;D bar is

Ed[f

riving voltage: 7.0 V; [Luminol]: 7.5 mM; Volume of GOD: 3 �L (1 U/�L). The error

CL intensity decreased. This phenomenon can be explained by the

ominating self-quenching effect at higher luminol concentrations50]. So, 7.5 mM luminol was selected as the optimal concentrationor further experiments.

the standard error from five independent experiments.

3.3. Analytical performance of the P-BPE-ECL system

H2O2 is often the focus of environmental, clinical, biological,and pharmaceutical studies. Moreover, it is used as an important

L. Chen et al. / Sensors and Actuators B 237 (2016) 308–317 315

Fig. 7. Proof-of-concept demonstration of high-throughput glucose detection using the proposed P-BPE-ECL method. (A) Schematic diagram of the device containing threesets of P-BPE-ECL units and one housing unit for avoiding the light disturbance from the near unit. (B, C) ECL images (i) and intensities (ii) with same glucose concentration( y. In pi

mopagtl(c(

1 mM) and with different glucose concentrations (0.1, 0.5 and 3.0 mM), respectivels the standard error from five independent experiments.

ediator in many analytical applications. Thus, the determinationf H2O2 is of great significance. Using the P-BPE-ECL system, theaper devices could be applied for quantification of H2O2 solutionst different concentrations. As depicted in Fig. 6A, ECL intensityradually increased as the H2O2 concentration increased from 5 �Mo 5000 �M. It has been indicated that the ECL intensity increasedinearly with the H2O2 concentration ranging from 0 to 100 �M

R2 = 0.9612) (Insert (a) in Fig. 6A), and with the logarithm of H2O2oncentration over the range of 200–5000 �M (R2 = 0.9824) (Insertb) in Fig. 6A), with relative standard deviations of 7.59%, 3.82%

anels (B) and (C), Driving voltage: 7.0 V; pH: 11.0; [Luminol]: 7.5 mM. The error bar

and 4.93% for 10, 100 and 1000 �M H2O2 (n = 5). In addition, thelimit of detection (LOD) was estimated to be 1.75 �M, where theLOD was defined as the lowest H2O2 concentration that produceda signal three standard deviations greater than the background.This LOD is comparable to that obtained in other BPE-ECL deviceswhose detection was performed in a commercial microcell withan ultraweak luminescence analyzer (0.8 �M) [19] or in a hybrid

PDMS-ITO glass microchip with a commercial chemiluminescenceimaging system (3 �M) [25], but is much lower than that reported

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n paper-based ECL devices using the detector of CCD (46.6 �M)40] or PMT (0.1 mM) [29].

.4. Glucose determination using P-BPE-ECL system

Over the last years, the glucose assay is often used to demon-trate the utility of paper microfluidic devices [43–45], and the PBSnd AU samples spiked with glucose are often applied to generatehe data [53]. Additionally, diabetes mellitus has become a globalpidemic due to its rapidly increasing incidence, and thus more andore attentions have been paid to the development of new glucoseonitoring devices. Hence, glucose was selected as a model target

o demonstrate the practical application of the P-BPE-ECL method.As we know, the determination of H2O2 is the basis of glucose

iosensing, and thus the presented P-BPE-ECL system could be usedor detection of glucose in PBS or AU sample. Note that the pH ofhe Na2CO3-NaHCO3 buffer has been further optimized for detec-ion of glucose. For glucose detection in PBS and AU, the optimalH values were 11.0 and 11.25, respectively (Fig. S2). It has beenhown that under optimal conditions, the ECL intensities were lin-arly correlated with the concentrations of glucose in PBS (Fig. 6B)r AU (Fig. 6C) samples, and the LOD values were estimated toe 0.017 mM (R2 = 0.9768, n = 5) and 0.030 mM (R2 = 0.9826, n = 5),espectively. These results show that the P-BPE-ECL system has thebility to detect different levels of glucose from different samples,nd to provide low detection limits. Although direct comparisons not possible at this stage because of the use of different meth-ds, it is worth noting that the presented detection levels are muchower than those typically obtained using EC, colorimetric, andhree-electrode ECL detection in paper devices [53].

.5. Evaluation of the potential of P-BPE-ECL system forigh-throughput screening

One advantage of using P-BPE-ECL with smartphone detectors that it has the potential to realize high-throughput detection.irstly, the preliminary experiments were performed to evaluatehether the light disturbance from the near unit existed in the

bsence of any light-interference prevention units. When the driv-ng voltage of 7.0 V was only applied to the paper channel “1”, theCL intensity on this channel was 0.8 × 103 counts (Fig. S3A); whilehe ECL intensity on the paper channel “1” was 0.85 × 103 counts ifhe similar driving voltage was imposed to each paper channel (Fig.3B). Obviously, the latter luminous value was about 6.3% higherhan the former one, indicating that the light disturbance did existn the proposed high-throughput detection system. Therefore, auitable light-interference prevention unit should be provided forhe detection of multiple samples.

In our case, a single paper device containing three sets of-BPE-ECL units was integrated with a well-designed housingnit that could be used to avoid the light disturbance fromhe near unit (Fig. 7A). This reusable housing unit mainly con-ists of four black, thin, rectangular plastic plates (Plates 1–4)Length × Height × Thickness: 30 mm × 20 mm × 1 mm), and theistance between the two adjacent plates is 6 mm and thus eachlate can be located vertically on the wax barrier of the paperevice. When the housing unit was placed onto the paper device,he paper channels “1”, “2” and “3” were separated from each other.t should be noted that when the height of plate was set to be 20 mm,he view of the camera could obtain the whole light emission fromach channel.

It has been shown that after three paper channels were sepa-

ated from each other by the housing unit and the driving voltagef 7.0 V was applied to the paper channel “1”, the ECL intensity ofhe paper channel “1” (Fig. S3C) was very similar to that obtained inig. S3A, indicating that the plates in the housing unit had an almost

[

ors B 237 (2016) 308–317

negligible effect on the self-luminescence on the paper channel. Incomparison of the results between Fig. 7B and Fig. S3C, the formerECL intensity from the paper channel “1” was only slightly higher(∼1.2%) than the latter one, indicating that light disturbance fromthe near unit had been eliminated to a great extent. The analyticalcase in Fig. 7B also showed that three paper channels had an obviousluminescence, and moreover had an almost negligible deviation. Inanother analytical case, the paper channels “1”–“3” were loadedwith 0.1, 0.5 and 3 mM glucose, respectively. After powering eachpair of driving electrodes, the “3” channel exhibited a strong ECLemission, while the “1” channel had a weaker ECL signal (Fig. 7C).These above results suggest that the P-BPE-ECL method has thepotential for high-throughput detection.

4. Conclusions

In this work, we realized the integration of paper microflu-idics, BPE-ECL, battery energy supply, and smartphone imagingto construct a handheld ECL system. Such an ECL system couldallow sensitive or high-throughput biochemical analysis to beeasily performed at the POC level. In our case, the luminol/H2O2-based P-BPE-ECL system exhibited a desirable analysis of H2O2,with acceptable dynamic range, detection sensitivity and detectionaccuracy. In addition, the P-BPE-ECL system could be well appliedfor highly-sensitive or high-throughput detection of glucose in dif-ferent samples. Looking to the future, the handheld P-BPE-ECLsystem may find broad applications, such as clinical diagnosis, envi-ronmental monitoring, and biomolecular interaction studies.

Acknowledgements

This research is supported by the National Natural Science Foun-dation of China (81571765), Guangzhou Science and TechnologyProgram (2014J4100030), and Guangdong Science and TechnologyProgram (2014A020212503; 2016A020215143).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2016.06.105.

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Biographies

Lu Chen received his B.S. degree in electronic and infor-mation engineering in 2013 from Hubei University ofTechnology. Currently, He studies in College of Biopho-tonics, South China Normal University as a postgraduatestudent.

Chunsun Zhang received his Ph.D. degree in 2006 fromUniversity of Science and Technology of China. He is cur-rently positioned as a professor at South China NormalUniversity. And, his research interests are focused on thedevelopment of microfluidic biosensor devices and ana-lytical tools.

Da Xing received the Doctor degree in Engineeringfrom Harbin Institute of Technology, China, in 1989and Ph.D. degree in Physics from University of Electro-Communications, Japan, in 1991. Since 1995, he has goneback to China and became the Director and professor of

Biometabolism.