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Sensors and Actuators B 248 (2017) 187–194

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo ur nal home page: www.elsev ier .com/ locate /snb

novel label-free homogeneous electrochemical immunosensorased on proximity hybridization-triggered isothermal exponentialmplification induced G-quadruplex formation

ong Qiana,c,1, Taotao Fana,c,1, Peng Wangd, Xing Zhangd, Jianjun Luod, Fuyi Zhouc,ao Yaoc, Xianjiu Liaob,∗, Yuanhong Li c, Fenglei Gaoc,∗

Jiangxi Province Key Laboratory of Polymer Micro/Nano Manufacturing and Devices, East China University of Technology, 330013, Nanchang, ChinaSchool of Pharmacy, Youjiang Medical University for Nationalities, 533000, Baise, ChinaJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medicalniversity, 221004, Xuzhou, ChinaThe Graduate School, Xuzhou Medical University, 221004, Xuzhou, China

r t i c l e i n f o

rticle history:eceived 24 November 2016eceived in revised form 24 March 2017ccepted 29 March 2017vailable online 31 March 2017

eywords:abel-freeemin

a b s t r a c t

A label-free homogeneous electrochemical immunosensor was developed for sensitive and selectivedetection of carcino-embryonic antigen (CEA) based on proximity hybridization triggered isothermalexponential amplification induced G-quadruplex formation. The presence of CEA promoted the forma-tion of a proximate complex via the proximity hybridization of the DNA strands labelled to affinity ligands,which unfolded the molecular beacon, the stem part of molecular beacon as a primer hybridize with thetemplate to initiate the isothermal exponential amplification process. Thus, with the electrochemicalindicator hemin selectively intercalated into the multiple G-quadruplexes, a significant electrochemi-cal signal drop is observed, which is dependent on the concentration of the target CEA. Thus, using this

omogeneousroximity hybridization

“signal-off” mode, a simple, label-free homogeneous electrochemical strategy for sensitive CEA assaywith a detection limit of 3.4 fg/mL is readily realized. Furthermore, this method also exhibits additionaladvantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobi-lization processes are avoided. Its high sensitivity, acceptable accuracy, and satisfactory versatility of

plica

analytes led to various ap

. Introduction

An immunoassay is considered as a powerful tool for dis-ase diagnosis [1–7], environmental monitoring [8,9], and foodafety [10,11]. Recently, electrochemical immunosensors haveained increasing attention and are considered to be one of theost promising methods in the quantitative detection of proteins

ecause of their specific advantages, such as low cost, excellentetection limits, fast response, and easy handling [12–15]. Vari-us electrochemical immunoassays based on labeling technologiesith electroactive substances, enzymes, and nanomaterials, have

een developed to amplify the tracing signal [16–19]. These electro-

hemical methods generally exhibit high detection sensitivity andigh signal to noise ratio. However, the aforementioned methodsre based on the heterogeneous assay that needs the immobiliza-

∗ Corresponding authors.E-mail addresses: lxj2006910@163.com (X. Liao), jsxzgfl@sina.com (F. Gao).

1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.snb.2017.03.152925-4005/© 2017 Elsevier B.V. All rights reserved.

tions in bioanalysis.© 2017 Elsevier B.V. All rights reserved.

tion of recognition element on the electrode [20,21]. Thus, thedevelopment of immobilization-free electrochemical strategies ishighly desirable.

Homogeneous electrochemical biosensor has drawn an increas-ing number of attentions in recent years, because the hybridizationbetween DNA and the recognition by the enzyme occur inthe homogeneous solution without the immobilization of anybiorecognition probe [22–26]. The signal outputs of these homo-geneous electrochemical biosensors are recorded via detecting thediffusion current of DNA labeled by electrochemical active com-pounds (methylene blue or ferrocene) in the solution [27–29].Although these homogeneous electrochemical strategies enabledlow detection limit, they still require modification of an elec-troactive substance at its terminal [30,31]. In order to avoid theunnecessary labeling process, it is desirable to design a convenientand efficient signal output electrochemical system for label-free

homogeneous detection.

In this paper, we construct a label-free homogeneouselectrochemical immunoassay based on proximity hybridization-

dx.doi.org/10.1016/j.snb.2017.03.152http://www.sciencedirect.com/science/journal/09254005http://www.elsevier.com/locate/snbhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.snb.2017.03.152&domain=pdfmailto:lxj2006910@163.commailto:jsxzgfl@sina.comdx.doi.org/10.1016/j.snb.2017.03.152

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riggered isothermal exponential amplification (IEA) for producingany G-quadruplex/hemin complexes. Hemin consists of an iron

III) ion held in a heterocyclic ring, known as a porphyrin [32]. Theemin use as an electron transfer medium based on the reversibleedox of Fe (III)/Fe(II) of hemin [33,34]. Unlike the most commonlysed redox mediator, hemin was not covalently modified at the endf the oligonucleotide but could formate of G-quadruplex/heminomplex. For example: Pu group have used hemin/G-quadruplexoncatamers as trace labels for sensitive detection of microRNAs35].

In this paper, using CEA as a model tumor marker, the tar-et CEA-induced proximity hybridization to trigger isothermalxponential amplification for sensitive electrochemical homoge-eous immunoassay was developed (Scheme 1). The presencef target CEA triggered the formation of sandwich proximitymmunocomplex, which unfolded the molecular beacon (MB). Sub-equently, this separated stem DNA as a signal primer to hybridizeith the template-DNA (T-DNA) and initiates IEA. The regener-

ted primer-template can continue to produce oligonucleotide-quadruplex in a manner of linear amplification. The productionf reporter oligonucleotide acts as a scaffold for the synthesis of-quadruplex/hemin. The intercalated hemin molecules are fur-

her prevented from reaching the electrode surface because of thelectrostatic repulsion between the negatively charged ITO elec-rode surface and the G-quadruplex. Thus, with fewer free hemin

olecules present in solution, a significant electrochemical currentrop is detected. Using this “signal-off” mode, facile and sensitiveomogeneous electrochemical detection of CEA is readily realized.

. Experimental section

.1. Reagents

Nt.BstNBI endonuclease and DNA polymerase were obtainedrom New England Biolabs. Deoxyribonucleotides (dNTPs) werebtained from Fermentas Biotechnology Co. Ltd (Canada). Prostate-pecific antigen (PSA), immunoglobulin G (IgG), platelet derivedrowth factor (PDGF), CA125, tris (2-carboxyethyl) phosphaneydro-chloride (TCEP), glucose (Glu), uric acid (UA), and dopamineDA), were obtained from Sigma-Aldrich Chem. Co. Water was puri-ed with a Milli-Q purification system (Branstead, USA) and usedhroughout the work. All chemicals used in this work were of ana-ytical grade. The buffers used in the study were HEPES buffer10 mM HEPES, 150 mM NaCl, pH 7.4) for target binding. The wash-ng buffer was PBS (50 mM Na2HPO4, 50 mM NaH2PO4, 0.1 M NaCl,H 7.5). DNA oligonucleotides used in this work were synthesizednd purified by Takara Biotechnology Co., Ltd. (Dalian, China).

DNA1: 5′-SH-TTTTTTTTTTTTTTTTGTGAGGGAACGGTCCTTG-3′

DNA2: 3′-SH-TTTTTTTTTTTTTTTTCACTCCCT-GCCAGATT-GGTAAC-5′

DNA3: 5′-ATTGCACCTGACGACGGTCAG-3′

G-quadruplex sequences: 5′-GGGTTAGGGTTAGGGTTAGGG-3′

Molecular beacon (MB): 3′-GTTACCTAATCTGGCTGCCAGGAACTACAGATTAGGTAAC-5′

Fluorophore-linker-MB:3′-FAM-GTTACCTAATCTGGCTGCCAGGATACAGATTAGGTAAC-BHQ-5′

Template DNA (T-DNA):3′-GTTACCTAATCTGTACCTCAGTT-TCCCAA

CCCGCCCTACCCAA-5′

.2. Apparatus

Differential pulse voltammetric (DPV) measurements were per-ormed using a CHI 660E electrochemical analyzer (Shanghai,hina). A three-electrode system was employed, with an indium tin

ors B 248 (2017) 187–194

oxide (ITO) electrode as the working electrode, an Ag/AgCl as thereference electrode, and a platinum wire as the auxiliary electrode.The ITO electrode was prepared as follows: first, ITO electrode wassequentially sonicated in an Alconox solution (8 g of Alconox perliter of water), acetone, and ultrapure water for 15 min each. Then,the ITO electrode was immersed into 1 mM NaOH solution for 5 hat room temperature and sonicated in ultrapure water for 15 min.After these cleaning procedures, a negatively charged working elec-trode surface was obtained. All the fluorescence measurementswere performed on a Hitachi F-7000 spectrofluorimeter (Hitachi,Japan). The excitation wavelength was 418 nm, and the spectraare recorded between 570 and 650 nm. The fluorescence emissionintensity was measured at 592 nm. Circular dichroism (CD) spec-tra were obtained on a JASCO J-815 spectrometer (Tokyo, Japan)at room temperature. CD spectra were recorded between 220 and350 nm with a 10 mm path length quartz cuvette. The scan ratewas set at 100 nm/min with a response time of 1 s and a bandwidthof 0.5 nm. The spectra were averaged over 3 scans. Zeta-potentialanalysis was performed on a Zetasizer (Nano-Z, Malvern, UK).

2.3. Preparation of DNA-labeled antibody

The DNA labeled antibodies (Ab-DNA) were synthesizedaccording to the previous work [36]. Briefly, anti-CEA anti-body (2 mg mL−1) was first reacted with a 20-fold molarexcess of sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) in PBS (55 mM phosphate, pH 7.4, 150 mMNaCl, 20 mM EDTA) for 2 h at room temperature. In parallel, 3 �Lof 100 �M DNA1 or DNA2 was reduced with 4 �L of 100 mMDTT in PBS for 1 h at 37 ◦C. Both products were purified byultrafiltration (10 000 MW cutoff membrane, Millipore), and thebuffer was changed to PBE (55 mM phosphate, pH 7.4, 150 mMNaCl, 5 mM EDTA). After the products were mixed to incubateovernight at 4 ◦C and the unreacted DNA was removed by ultra-filtration (100,000 MW cutoff membrane, Millipore), the Ab-DNAwas obtained.

2.4. Measurement procedure

The isothermal amplification reaction was carried out in 25 �Lof total reaction solution consisting of 10 �L of 1 �M Ab1-DNA1and Ab2-DNA2, 5 �L of various concentrations of CEA, 5 �L of 1 �MMB, 5 �L of 1 �M T-DNA, 5U DNA polymerase, 3.0 mM dNTP and6U nicking enzyme. The one-pot reaction system was incubatedat 37 ◦C for 50 min in a mixed buffer of NEBuffer 2 (50 mM NaCl,10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) and CutSmartBuffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM mag-nesium acetate, 100 mg/mL BSA, pH 7.9). The reaction solution and5 �L hemin (12 �M) were dropped onto the ITO for electrochemi-cal detection, which could adequately cover the electrode workingarea. DPV from 50 to 750 mV (vs Ag/AgCl) with pulse amplitudeof 50 mV and a pulse width of 200 ms was recorded in the above-mentioned reaction mixtures.

3. Results and discussion

3.1. Feasibility of the electrochemical immunoassay

The proof-of-concept experiments were carried out to investi-gate the feasibility of the proposed strategy for CEA assay. To verifythat proximity hybridization could open MB, hybridization testsusing free DNA strands in solution were performed. A fluorophore

and a quencher were modified at both ends of the MB. As shown inFig. 1, by itself or in the absence of target CEA, a weak fluorescenceemission was observed (curve a) since the fluorescence of FAM wasquenched by the black hole quencher (BHQ). In addition, for the

Y. Qian et al. / Sensors and Actuators B 248 (2017) 187–194 189

Scheme 1. Schematic representation of label-free electrochemical homogeneous immunosensor strategy based on proximity hybridization regulated IEA induced G-quadruplex formation.

F re-linC ntatioT ng mL

siaotrscat

ticht

ig. 1. (A) Fluorescence spectra of (a) fluorophore-linker-MB (1 �M); (b) fluorophoEA (1 ng mL−1); Ab-DNA1 (1 �M) and Ab-DNA1 (1 �M); Inset: Schematic represe-DNA, polymerase, endonuclease and dNTPs, (b) +1 ng mL−1 target CEA; (b) (a) + 1

olution containing Ab-DNA1 and Ab-DNA2 only, the fluorescencentensity was not changed (curve b) compare to the blank (curve), indicating that Ab-DNA1 and Ab-DNA2 could not open the loopf MB owing to the binding of the complementary sequences athe ends. However, upon addition of target CEA, an enhanced fluo-escence peak was observed (curve c), indicating that CEA triggeredufficient proximity to undergo hybridization and form a proximityomplex, which led to a sequence to subsequently unfold the MBnd separate FAM from its quencher BHQ and therefore reducinghe quenching effect and producing fluorescence.

The IEA reaction was confirmed with circular dichroism spec-ra analysis (Fig. 1B). The appeared G-quadruplex is the core of thesothermal exponential amplification. CD spectroscopy was used to

onfirm the formation of a G-quadruplex, because G-quadruplexesave the characteristic CD spectra. It is known that a parallel struc-ure has a positive peak around � = 260 nm and a negative minimum

ker-MB (1 �M), Ab-DNA1 (1 �M) and Ab-DNA1 (1 �M); (c) fluorophore-linker-MB,n of fluorescence verification strategy. (B) CD spectra of (a) Ab-DNA1, Ab-DNA2,

−1 target CEA, (c) (b) after reaction for 50 min; and (d) (c) + hemin.

around � = 240 nm. The 260 nm CD band in parallel G-quadruplexDNA has been attributed to the change in glycosidic bond rota-tion and consequently to the stacking arrangement of bases whilethe 240 nm band reflects on right handed helicity of DNA[37]. Asshown in Fig. 1B, the CD spectrum is of relatively low amplitude inthe absence of CEA (curve a), which suggests that no G-quadruplexstructure. When the solution incubated with CEA, the spectrumdisplayed a positive peak at longer wavelength near 260 nm anda negative peak near 240 nm, which is consistent with the par-allel G-quadruplex structure (curve b) produced in the cycling ofIEA via the polymerization and strand-scission process. The neg-ative and positive peaks peak intensity become very larger after50 min of reaction time (curve c), the obtained IEA product was

thousands of G-quadruplex sequences. The dominant conformationof G-quadruplex remained unchanged before and after the addi-tion of hemin, meaning heimin did not affect the main structure of

190 Y. Qian et al. / Sensors and Actuators B 248 (2017) 187–194

Fig. 2. (A) DPV curves for the sensor in the presence of (a) 12 �M hemin; (b) 12 �M hemin + 10 nM DNA3; (c) 12 �M hemin + 0.1 nM G-quadruplex sequences; (d) 12 �Mh ence op action

Gaawo

fttnvbotvrwho(co(etTmrwpAtbtabnntdtc

emin + 10 nM G-quadruplex sequences. (B) DPV curves for the sensor in the presolymerase, endonuclease and dNTPs, (c) (b) + 1 ng mL−1 target CEA; (d) (c) after re

-quadruplex. However, the ellipticities of characteristic negativend positive peaks were a little higher than those of G-quadruplexlone (curve d). It should be attributed to the interaction of heminith G-quadruplex, which could stabilize and favor the formation

f G-quadruplex [38].It is the basis of our new signal-amplified imunosensor that a

ree hemin produces a much higher electrochemical signal of heminhan hemin locked into the G-quadruplex, as a result of the elec-rostatic repulsion between the negatively charged DNA and theegatively charged electrode. Zeta-potential analysis was used toerify the electrostatic repulsion. Zeta potential was determinedy the mobility of ions in an electric field and the mobility dependsn the substance charge [39]. Fig. S1 displays zeta potential dis-ributions of hemin, G-quadruplex/hemin and ITO electrode at pHalues of 7.4. Its zeta potential was −0.2 mV, −40 mV, and −43 mVespectively. We have proved the diffusion current of bound heminith G-quadruplex decreases significantly as compared to that ofemin (curve a). As shown in Fig. 2A, we observed the redox currentf hemin decreased in the presence of a G-quadruplex sequencecurve c). Also, the diffusion current of hemin was related to theoncentration of G-quadruplex. The higher was the concentrationf G-quadruplex, the lower was the current of hemin obtainedcurve d). However, the current remained unchanged in the pres-nce of the other DNA sequences (curve b). The result indicated thathere exist strong interaction between G-quadruplex and hemin.his result indicated that as a weak charged hemin molecules canove onto the surfaces of ITO. The reduction of current response

esults from the electrostatic repulsion of G-quadruplex/heminith ITO. As shown in Fig. 2B (curve a), a readily detectable DPVeak at around −0.4 V could be observed in the presence of hemin,b-DNA1, and Ab-DNA2. This peak could be attributed to the elec-

rochemical oxidation of the hemin because hemin molecules cane absorbed onto the surfaces of ITO through electrostatic. Withhe mixture of hemin, Ab-DNA1, Ab-DNA2, T-DNA, polymerasend dNTPs, the electrochemical signal barely unchanged (curve b),ecause in the absence of the target (CEA), the IEA reaction didot occur, resulting in no hemin/G-quadruplex produced and thuso electrochemical signal changed. In the presence of target CEA,

he mixture of hemin, Ab-DNA1, Ab-DNA2, T-DNA, polymerase andNTPs, it was found, as the DPV scanning results show in curve c,hat the peak signal of hemin is remarkably reduced during theourse of IEA, compared to that with no target input. The peak

f (a) blank (hemin, Ab-DNA1, Ab-DNA2); (b) hemin, Ab-DNA1, Ab-DNA2, T-DNA, for 50 min.

current becomes small after 50 min of reaction time (curve d), theobtained IEA product was thousands of G-quadruplex sequencesfor hybridization with hemin, which supports well the signal-offmechanism as a result of the electrostatic repulsion between thehemin/G-quadruplex and the negatively charged electrode. Thus,the aforementioned results clearly demonstrated the feasibility ofour strategy for CEA assay.

3.2. Optimization of detection conditions

The reaction conditions have important effect on the sens-ing process, such as the amount of hemin reporters, polymerase,endonuclease, dNTPs, and incubation time of IEA on the electro-chemical signal. Therefore, the reaction conditions were optimizedsuccessively in order to achieve the best signal-to-noise level. Theinitial concentration of hemin plays an important role in the per-formance of the biosensor. An excessive amount of hemin canlead to a high background signal, and lower concentration willcause the very low detection signal, which affects the sensitivityof the proposed sensor. So the effect of hemin concentration hadbeen optimized first. As shown in Fig. 3A, the DPV peak currentchange �ip (i.e., the difference between the DPV peak current inthe presence of the target and that of the blank) exhibited a sharpincrease as the hemin concentration increased from 1 �M to 14 �M.In our strategy, �ip needs to be big enough to indicate the cur-rent change induced by the target. Therefore, 12 �M hemin hasbeen chosen as the optimized condition. As shown in Fig. 3B–D,the current change increased with the increasing amount of poly-merase, endonuclease and dNTPs, and then trended a maximumvalue, indicating a saturated amplification. The optimal amount ofpolymerase, endonuclease and dNTPs were selected at 5.0 U, 6.0 Uand 3.0 mM, respectively. The reaction time was another impor-tant parameter affecting the analytical performance. It was clearthat the current response increased with the increase of reactiontime and tended to a maximum value at 50 min (Fig. 3E). Hence,the optimum reaction time in this work was 50 min.

The detection mechanism of the electrochemical immunoassayrelied on simultaneous recognition of target protein by two probes,

which brought DNA1 and DNA2 in proximity to hybridizationand subsequently unfold MB. Thus, the number of complemen-tary bases between DNA1 and DNA2, DNA1 and MB, DNA2 andMB should be firstly optimized. At low number of complementary

Y. Qian et al. / Sensors and Actuators B 248 (2017) 187–194 191

F he amr optim

bwbF671e

ig. 3. Dependence of the DPV peak current change for 1 pg mL−1 target CEA on teaction time of IEA. In each optimization experiment, other parameters were their

ases, DNA1 and DNA2 could not hybrid even in close proximityith the help of target, while at high number of complementary

ases, DNA1 and DNA2 could self-hybrid and produce a large noise.ig. S1 shows the signal-to-noise of the system using DNA2 with 5,

, 7, 8, 9 and 10 complementary bases (bp) to DNA1; DNA1 with, 8, 9, 10, 11, 12 complementary bases (bp) to MB, DNA2 with 12,3, 14, 15, 16 and 17 complementary bases (bp) to MB. In the pres-nce of CEA (0.1 ng/mL), the signal-to-noise ratio increased quickly

ounts of (A) hemin, and (B) dNTPs, (C) the polymerase, (D) endonuclease, (E) theized values. Error bars represent standard deviations of three parallel experiments.

with the increasing number of complementary bases from 5 to 8 bp(Fig. S2A), 7 to 10 bp (Fig. S2B), 12 to 15 bp (Fig. S2C), and thendecreased. According to the maximum signal-to-noise ratio, DNA2with 8-bp complementary to DNA1, DNA1 with 10-bp complemen-

tary to MB, DNA2 with 15-bp complementary to MB, were chosenfor the subsequent experiments.

192 Y. Qian et al. / Sensors and Actuators B 248 (2017) 187–194

Fig. 4. (A) DPV responses of homogeneous electrochemical immunoassay of CEA at blankagainst the concentration of CEA. Inset: the linear plot of the DPV peak current changedeviations of three parallel experiments.

Table 1Comparing of the proposed method with other immunoassays.

Analytical method Detection limit Linear range Refs.

Electrochemical 1.3 pg/mL 0.002–100 ng/mL [23]Electrochemical 153 pg/mL 200 pg/mL–10 �g/mL [22]

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Electrochemical 6 pg/mL 0.01–3.5 ng/mL [24]Electrochemical 3.4 fg/mL 10 fg/mL–1 ng/mL This work

.3. Assay performance

Under the optimal conditions, the DPV peak current ofmmunosensor for CEA measurement gradually decreased withhe increase of concentrations of CEA (Fig. 4A). The relationshipetween the DPV peak current change (�ip) and the concentrationf CEA is shown in Fig. 4B. Obviously, the concentration of CEA inhe solution from 10 fg/mL to 1 ng/mL could be detected by theroposed strategy. When the DPV peak current change (�ip) werelotted against the logarithm of the analyte concentrations (inset ofig. 4B), the resulting standard curve showed a linear relationship inhe range from 10 fg/mL to 1 ng/mL. The linear regression equationas expressed as I (�A) = 1.941 + 0.134 lg C (g/mL) with a correla-

ion coefficient of 0.9905. The limit of detection at a signal-to-noiseatio of 3 was estimated to be 3.4 fg/mL, which is much lowerhan other electrochemical homogeneous immunoassay methodseported previously (Table 1). Compared with these electrochemi-al immunosensor methods [22–24], this detection process do noteed to synthesize the nanocomposite probe and do not display aertain degree of nonspecific release. In addition, the whole processs only one step without the requirement of time-consuming sepa-ation and washing steps. The above results indicated the successfulchievement of the electrochemical amplified CEA detection, whichhould be attributed to the factor: the linearity amplification of IEArocess, which made many of hemin far away from the surface of

TO.To further examine the specificity of the electrochemical

ethod toward the target CEA, the system was used for challeng-ng prostate-specific antigen (PSA), PDGF, CA125, and IgG. As seen

rom Fig. 5A, the currents obtained from PSA, PDGF, CA125, andgG alone were almost the same as the background signal. More-ver, their mixture with CEA did not cause a significant increase ofhe electrochemical signals relative to the pure target analyte. To

(a), 10−14–10−8 g mL−1 (from b to h), and (B) the DPV peak current change plotted versus the logarithm of the concentration of CEA. Error bars represent standard

investigate the interfering effects of sample matrix components onthe analytical properties of the developed method, several possiblecomponents in the serum, such as Na+, Mg2+, Cl−, glucose (Glu),uric acid (UA), and dopamine (DA), were added into the incuba-tion solution containing 1 pg mL−1 CEA, respectively. As shown inFig. 5B, these interfering ions did not almost affect the significantchange in the electrochemical signal relative to target CEA alone.The high specificity and anti-interference of the developed methodmight be ascribed to the specific antigen−antibody reaction and thestrong proximity hybridization between Ab-DNA1 and Ab-DNA2.

3.4. Stability and reproducibility of the immunosensor

As an important property of the fabricated homogeneousimmunosensor, the storage stability is necessary to be investigated.When the homogeneous immunosensor was stored in the refrig-erator at 4 ◦C and measured every 1 week. As shown in Fig. S3, noobvious change was found during the first week and the responsechanged less than 1.6% of the original current. After storing for2 weeks, it maintained 95.5% of the initial response, suggest-ing quite satisfactory storage stability. The reproducibility of theimmunosensor was studied by inter- and intra-assay coefficients ofvariation according to the following methods: the interassay wasevaluated by analyzing the CEA using five equally immunosensorsunder the same conditions, where all electrodes showed approxi-mate electrochemical responses and a RSD of 4.6% were obtained.The intra-assay was estimated by analyzing the CEA level for fivetimes using the same prepared immunosensor, and a relative stan-dard deviation (RSD) of 3.7% was acquired (the data in supportinginformation), which indicates an acceptable reproducibility of theas-proposed strategy.

3.5. Application in detection of serum tumor marker

To evaluate the analytical reliability and application potential ofthe proposed method, it was used to detect CEA in clinical serumsamples, and the assay results were compared with the refer-

ence values from the commercial electrochemiluminescent testing.When the levels of tumor markers were over calibration ranges,serum samples were appropriately diluted with 0.01 M PBS (pH 7.4)prior to the assay. The results shown in Table 2 showed an accept-

Y. Qian et al. / Sensors and Actuators B 248 (2017) 187–194 193

Fig. 5. (A) The specificity of the homogeneous immunosensor against (a) blank, (b) 0.1 1 pg mL−1 CEA+ PSA; (f) 1 pg mL−1 CEA+ PDGF; (g) 1 pg mL−1 CEA+ CA125; (i) 1 pg mL−1 CE

Table 2Assay results of clinical serum samples using the proposed and reference methods.

Sample Proposed method(ng mL−1)

Reference method(ng mL−1)

Relative error (%)

1 0.024 0.026 −7.692 0.33 0.31 6.45

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ble agreement with relative errors of less than 7.69%, indicatingood accuracy of the proposed method for the detection of clinicalamples.

. Conclusions

In summary, we have developed a simple, label-free, homoge-eous electrochemical biosensing strategy for sensitive detectionf CEA based on proximity hybridization-triggered isothermalxponential amplification. This label-free design does not requireny chemical modification for DNA or sophisticated equipments,hich offers the advantages of simplicity and cost efficiency.otably, taking advantage of exponential amplification efficiencyf IEA, the proposed method exhibits excellent sensitivity with

detection limit of 3.4 fg/mL. Owing to the requirement of twoimultaneous binding events for a single target molecule, the assayxhibits high specificity, Furthermore, it is a fast and easy-to-usemmobilization-free method that was carried out in the homo-eneous solution; thus, complex modification or immobilizationrocedures are avoided. The assay can be readily extended to otheriological molecules with available affinity ligands to form theroximate complex and thus possesses great potential for point-f-care disease diagnostics.

cknowledgments

This work was supported by the National Natural Scienceoundation of China (21405130, 21565002, 21665026), Most Foun-ation of Jiangxi Province (20152ACB21018), Excellent Talents ofuzhou Medical College (D2014007), Qing Lan Project.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.snb.2017.03.152.

[

ng mL−1 PSA, (c) 0.1 ng mL−1 PDGF, (d) 0.1 ng mL−1 CA125, (e) 0.1 ng mL−1 IgG, (e)A+ IgG; (j) 1 pg mL−1 CEA; (B) Sample matrix interfering effect.

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research interest is in the area of biosensors.

Fenglei Gao received his PhD degree in analytical chemistry from Nanjing Univer-sity, China in 2013, respectively. Now he is an associate professor of Xuzhou Medical

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Biographies

Yong Qian received his PhD degree in polymer science and engineering from NanjingUniversity, China in 2009, respectively. Now he is an associate professor of East ChinaInstitute of Technology, China. His research interest is in the area of electrochemicalbiosensors.

Taotao Fan entered the MS course in 2015, majored in chemistry.

Peng Wang entered the MS course in 2014, majored in neurosurgery.

Xing Zhang entered the MS course in 2016, majored in sports medicine.

Jianjun Luo entered the MS course in 2016, majored in sports medicine.

Fuyi Zhou entered the MS course in 2014, majored in pharmaceutical analysis.

Yao Yao entered the MS course in 2016, majored in pharmaceutical analysis.

Xianjiu Liao received his PhD degree in analytical chemistry from Nanjing Uni-versity, China in 2015. Now he is a lecturer of Youjiang Medical University forNationalities. His research interest is in the area of biosensors.

Yuanhong Li received his PhD degree from Nanjing Agricultural University, Chinain 2013, respectively. Now he is a lecturer of Xuzhou Medical University, China. His

University, China. His research interest is in the area of biosensors.

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