ORIGINAL PAPER

Ultrasensitive and selective voltammetricaptasensor for dopamine based on a conducting polymernanocomposite doped with graphene oxide

WentingWang &WeiWang & Jason J. Davis &Xiliang Luo

Received: 31 August 2014 /Accepted: 14 November 2014# Springer-Verlag Wien 2014

Abstract We describe an aptasensor for the determination ofdopamine in human serum and with ultrahigh sensitivity andselectivity. The sensor is based on a nanocomposite consistingof reduced graphene oxide (rGO) and the conducting polymerpoly(3,4-ethylenedioxythiophene) (PEDOT). The PEDOT/rGO interface was prepared by electrochemical polymeriza-tion of EDOT using graphene oxide as the dopant which islater electrochemically reduced to form rGO. Subsequentcovalent modification of the high surface area composite witha selective aptamer enables highly sensitive and selectivedetection by differential pulse voltammetry. The calibrationplot established at a working voltage of 160 mV displays alinear response in the 1 pM to 160 nM concentration range andan unprecedented detection limit of 78 fM. The sensor is fairlyselective in not responding to common interferents, and isreusable after regeneration with a 7 M solution of urea. Itwas successfully applied to (spiked) serum samples and gaverecoveries ranging from 98.3 to 100.7 %.

Keywords Aptamer . Conducting polymer . Dopamine .

Graphene oxide . Poly(3,4-ethylenedioxythiophene)

Introduction

Abnormal levels of brain dopamine (DA) are implicated in thepathology of a number of increasingly prevalent psychiatricstates and neurodegenerative diseases. A quantitative, conve-nient, sensitive and selective assessment of dopamine is thusof considerable value. DA is a member of the catecholamineneurotransmitter family playing a significant role in commu-nication within the central nervous system [1]. Its dysfunctionhas been implicated in multiple diseases such as Parkinson’sand Huntington’s diseases, Alzheimer’s disease, Tourette syn-drome, schizophrenia, psychosis, and drug addiction [2, 3].Additionally, DA has been investigated as a peripheral bio-marker for the diagnosis of Parkinson’s disease [4]. There isenormous value in developing selective, sensitive and low-cost methods for its assay, and a broad range of methodolo-gies, including those based on chromatography [5], spectros-copy (optical) [6], electrophoresis [7], fluorescence [8], andcolorimetric analysis [9] have, accordingly, been reported.The cost and multistep nature typical of many of theseapproaches has highlighted the value that appropriatelabel free electroanalyses have. Electrochemical methodsthat take advantage of the redox activity of DA [2, 10,11] have received significant attention and bring sim-plicity, sensitivity, speed and low cost to the table.There remain, however, profound challenges. Firstly,the clinically relevant levels of DA present in manybiological samples are low. Secondly, the large amountof electroactive endogenous interferents (most notableuric acid and ascorbic acid) [12] makes selective detec-tion, with any realistic sample, difficult. Compoundingthis further is the electrode-fouling nature of the oxida-tion product of DA (progressively reducing assay per-formance) [13].

Aptamers are artificial functional oligonucleic acids de-rived from random sequence combinatorial libraries through

W. Wang :W. Wang :X. Luo (*)Key Laboratory of Sensor Analysis of Tumor Marker, Ministry ofEducation, College of Chemistry and Molecular Engineering,Qingdao University of Science and Technology, Qingdao 266042,Chinae-mail: xiliangluo@hotmail.com

J. J. Davis (*)Department of Chemistry, University of Oxford, Oxford OX1 3QZ,UKe-mail: Jason.davis@chem.ox.ac.uk

Microchim ActaDOI 10.1007/s00604-014-1418-z

a systematic evolution of ligands by exponential enrichment(SELEX), and can be reproducibly synthesized in a largequantity in vitro [14, 15]. Theoretically, aptamers are capableof binding to a remarkably broad range of cognate targetmolecules with, in many cases, high affinity and specificity[16–18]. They do this whilst remaining readily synthesized,stored and modified [19, 20]. They are more conformationallyrobust than antibodies or enzymes, and can undergo multipledenaturation/regeneration cycles (facilitating capture and re-lease of target molecules) without loss of efficacy in reusableapplications [21, 22]. With these characteristics, the exponen-tial growth in their application within electrochemical sensorformats is understandable [23, 24].

In the past few years, a limited number of differentaptasensors for the detection of DA have been reported. Forexample, Zheng et al. have developed a colorimetric biosensorfor the detection of DAwith a DNA aptamer as the recognitionelement, and it showed an analytical linear range from 5.4×10−7 to 5.4×10−6 M and a limit of detection of 3.6×10−7 M[25]. Electrochemical aptasensors for DA have been fabricat-ed based on both RNA aptamer [26] and DNA aptamer [27].The former was developed based on the immobilization ofRNA aptamer on a cysteamine modified gold electrode andthe direct oxidation of DA on the gold electrode, and itallowed a selective detection of DA within the 100 nMto 5 μM range [26]. The latter was developed usingDNA aptamer and it exhibited a linear response to DAin the range 0.007–90 nM and a limit of detection of1.98 pM [27], which is much more sensitive than theformer. However, this DNA aptasensor was based ondrop-coated graphene-polyaniline nanocomposites, and itrequired the addition of a redox probe [Fe(CN)6]

3-/4- toenhance the sensitivity, which makes the detection lessreproducible/reliable and more complicated.

In previous work, our group has reported a chemicalsensor for DA based on a electrode modified with theelectrodeposited nanocomposite of conducting polymerpoly (3,4-ethylenedioxythiophene) (PEDOT) doped withreduced graphene oxide (rGO), and it could detect DAwithin a wide linear range from 0.1 to 175 μM inbuffered aqueous solution due to the electrocatalyticactivity of the PEDOT/rGO nanocomposites towards tothe oxidation of DA [28]. In this work, we have inte-grated aptamers into the PEDOT/rGO nanocompositemodified electrode interface in order to promote levelsof selectivity and sensitivity that are both unprecedentedand enable robust DA quantification in real biologicalsamples. The marriage of the high specificity of theaptamer and the excellent electrocatalytic activity ofthe nanocomposite towards the oxidation of DA bringswith it the capability of electrochemical DA detection ina label free and reagentless (without redox probe) man-ner with ultrahigh sensitivity and selectivity.

Experimental

Chemicals and apparatus

The 58-mer dopamine-binding aptamer derived in SELEXand functionalized with amine group at the 5′ end (NH2-5′-GTC TCT GTG TGC GCC AGA GAA CAC TGG GGCAGATAT GGG CCA GCA CAG AAT GAGGCC C-3′) wassynthesized by Sangon Biotechnology Co., Ltd. (Shanghai,China, http://www.sangon.com), purified by high-performance liquid chromatography and used as received.Aptamer stock solution was prepared by dissolving the pur-chased aptamer in phosphate buffered saline (PBS, 0.2 M, pH7.4) and stored at 4 °C before use. Dopamine hydrochloride,AA, UA, glucose, N- (3-Dimethylaminopropyl)-N’-ethylcarbodiimid (EDC), N-hydroxysuccinimide (NHS) andethanolamine were purchased from Aladdin Reagents(Shanghai, China, http://www.aladdin-reagent.com/).Graphene oxide was purchased from Nanjing Xian FengNanomaterials Technology Co., Ltd. (Nanjing, China, http://www.xfnano.com/). 3,4-ethylenedioxythiophene (EDOT)was obtained from Aladdin Reagents (Shanghai, China,http://www.aladdin-reagent.com/). Human serum sampleswere provided by Chengyang People’s Hospital in Qingdao,China. All other reagents were of analytical reagent grade andused as received. Millipore water from a Milli-Q water puri-fying system was used throughout. All experiments wereperformed at ambient room temperature.

Electrochemical experiments, including electrochemicalimpedance spectroscopy, differential pulse voltammetry andcyclic voltammetry, were carried out with a CHI 760D elec-trochemical workstation (Shanghai Chenhua Instrument Co.Ltd., China). All experiments were performed in a conven-tional three-electrode system consisting of a platinum wirecounter electrode, an Ag/AgCl (3 M KCl) reference elec-trode and a glassy carbon working electrode (diameter3.0 mm). Field emission scanning electron microscope(SEM) was performed with a JEOL JSM-7500 F SEMinstrument (Hitachi High-Technology Co., Ltd., Japan).Differential pulse voltammetric measurements were re-corded from −0.2 to 0.5 V, and the pulse amplitude, thepulse width and the pulse period were set as 50 mV, 0.1and 0.2 s, respectively. The electrochemical impedancespectroscopy measurements were recorded in 5.0 mM[Fe(CN)6]

3-/4- solution containing 0.1 M KCl. The am-plitude of the applied sine wave potential was 5 mV, thefrequency range was from 1 Hz to 100 kHz and theapplied potential was 0.25 V.

Fabrication of the aptasensor

The schematic illustration of the aptasensor fabrication isshown in Scheme 1. Briefly, GCEs were polished, cleaned

W. Wang et al.

and electrochemically pretreated according to a previous re-port [29]. Following pretreatment, PEDOT/GO nanocompos-ite films were electrochemically deposited onto GCEs from apolymerization solution of water containing 2.0 mg mL−1 GOand 0.02 M EDOT, using cyclic voltammetry with the poten-tial scanning between −0.2 and 1.2 Vat a scan rate of 100 mVs−1 for 13 cycles. The PEDOT/rGO nanocomposite films werethen obtained through electrochemical reduction of thePEDOT/GO nanocomposite films in PBS (0.2 M, pH 7.4)by applying a potential of −0.9 V for 600 s [28]. GCEsmodified with the nanocomposite films before and after elec-trochemical reduction were denoted as PEDOT/GO/GCE andPEDOT/rGO/GCE, respectively. For the construction ofaptasensors, the PEDOT/rGO films were firstly treatedwith a solution containing 0.4 M EDC and 0.1 M NHSfor 30 min to activate the terminal carboxyl groups ofrGO on the electrode interface. The activated PEDOT/rGO/GCEs were then incubated in 1.0 M amine-functionalized aptamer solution (0.2 M PBS, pH 7.4)for 3 h to allow the covalent attachment of aptamer tothe electrode surface. To ensure all of the EDC/NHSactivated carboxyl groups had reacted, the modifiedelectrodes (after aptamer immobilization) were finallyimmersed in 1.0 M ethanolamine (pH 8.5) for 30 minto quench the rest activated carboxyl groups [30].

Electrochemical detection of DA

For the DPV detection of DA (in buffer or human serum), theaptasensor was incubated in the solution or sample containingDA for 60 min to ensure effective binding of DA to theimmobilized aptamer on the electrode surface. The incubatedaptasensor was rinsed thoroughly with PBS to remove anyphysically adsorbed DA or other substances, and then theaptamer associated DAwas electrochemically detected usingDPV in pure PBS (0.2 M, pH 7.4). For the purpose of exam-ining reusability of the aptasensor, the used aptasensor was

Fig. 1 A SEM image of the PEDOT/rGO nanocomposite film; B SEMimage of the PEDOT/rGO nanocomposite film modified with aptamer;CNyquist plots of the EIS for the bare GCE (a), the PEDOT/GO/GCE (b),the PEDOT/rGO/GCE (c), the aptamer modified PEDOT/rGO/GCE (d)and the aptamer modified PEDOT/rGO/GCEwith binded DA (e). Inset isthe Randles equivalent circuit model

Scheme 1 Schematic illustration of the preparation and sensing of theaptasensor. Graphene oxide and EDOT was electrochemically depositedonto a glassy carbon electrode to form a composite of PEDOT/GO, and itwas further electrochemically reduced to PEDOT/rGO (carboxyl groupof rGO was retained); Aptamer functionalized with amine group wasattached to the PEDOT/rGO nanocomposite film through the reactionbetween the amine and carboxyl groups; Dopamine was selectivelycaptured by the aptamer, and the collected dopamine was thenelectrochemically detected after washing

Ultrasensitive dopamine aptasensor

immersed in 7.0 M urea for 15 min, and then thorough-ly rinsed with PBS (0.2 M, pH 7.4). The regeneratedaptasensor was then used for DA determination similar-ly as above.

Results and discussion

Characterization of the modified electrodes

The preparation and biosensing of the aptasensor was illus-trated in Scheme 1. PEDOT/rGO nanocomposite films wereelectrodeposited onto bare glassy carbon electrodes (GCE)f r om an aqueou s s o l u t i o n o f monome r ( 3 , 4 -ethylenedioxythiophene, EDOT) and graphene oxide (GO)nanosheets, followed by electrochemical reduction of GO torGO [28]. The morphology and microstructure of the resultingfilm was characterized by scanning electron microscopy(SEM) (Fig. 1A), where rough wrinkles, consisting ofsub-micron and sheet-like features are evident (the latterlikely to comprise rGO nanosheets embedded in thePEDOT matrix). The carboxyl rich nature of thePEDOT/rGO film facilitates easy aptamer attachmentthrough terminal amine groups. The morphology of theaptamer functionalized PEDOT/rGO film (shown inFig. 1B, predictably showing little difference from thatof the unmodified film) is retained through thiscoupling.

Electrochemical impedance spectroscopy (EIS) presents auseful means of characterizing the stepwise modification of anelectrode surface [31, 32]. Nyquist impedance plots include asemicircle portion at higher frequencies related to the charge-transfer process and a linear portion at lower frequenciescorresponding to any diffusion limited process. The formercan be quantified through the semicircle diameter as thecharge-transfer resistance (Rct) of the electrode, when fittedusing a standard Randles equivalent circuit (inset in Fig. 1C)[33, 34]. Predictably, Rct decreases after the deposition of theconducting polymer nanocomposite (PEDOT/GO) film, andfurther falls when the PEDOT/GO is electrochemically re-duced to PEDOT/rGO (curves a, b and c of Fig. 1C). Thelatter may be attributed to the fact that GO in the nanocom-posite is reduced to the more conductive rGO [35].Subsequent aptamer immobilization increases Rct (curved, Fig. 1C) owing to the fact that its negatively chargedphosphate backbone characteristically repels the nega-tively charged [Fe(CN)6]

3-/4- probe. Interestingly, expo-sure of the so modified interface to DA leads to afurther increase in Rct (curve e, Fig. 1C), potentiallydue to the conformational change of the aptamer afterits association with DA. The increased spatial restriction

of a folded aptamer-target conjugate has been noted inother work [27].

Response of DA at different electrodes

In this work, DA quantification at the prepared aptasensorinterfaces was carried out by incubation in DA containingsamples for a finite period of time, prior to washing to removeany nonspecific surface association and analysis by differen-tial pulse voltammetry (DPV) in phosphate buffered saline(PBS, 0.2 M, pH 7.4). As shown in Fig. 2 (curves e and f),notable DPV responses are observed after incubation in1.0 nM DA solution. Significantly, almost no amperometricresponse is observed with bare (curves a and b, Fig. 2) orPEDOT/rGO modified electrodes (curves c and d, Fig. 2) inthe absence of aptamer. This result clearly indicates thataptamer can selectively bind DA to the electrode surface,which can separate DA (after washing) from the sample andthus prevent possible interferences.

Fig. 2 DPV curves for the bare GCE (a, b), the PEDOT/rGO/GCE (c, d)and the aptasensor (e, f) before (a, c, e) and after (b, d, f) incubation in1.0 nM DA solution for 60 min. The DPV curves were measured in PBS(0.2 M, pH 7.4)

Fig. 3 The effect of incubation time on the DPV current response of theaptasensor towards the detection of 10 nM DA. The DPV workingvoltage is ranging from −0.2 to 0.5 V

W. Wang et al.

Aptasensor incubation time

The role of the confined aptamer is to selectively recruit DA atthe electrode interfaces in a manner which both“preconcentrates” it and enables its direct determination byDPV. The optimal timeframe of the assay requires, then, aconsideration of both diffusion from solution and accumula-tion at the aptamer interface prior to electroanalysis. As isshown in Fig. 3, aptasensor response gradually increases withincubation time up to 60 min, prior to a levelling off at~70 min. For all assays, thereafter, incubation times weremaintained at the apparent 60 min optimum.

Sensing performance of the aptasensor

The ability of these interfaces to quantify DA was surveyedacross a broad solution concentration range (Fig. 4). The insetof Fig. 4 shows the linear range of such assessments (0.001 to160 nM, with a linear regression equation asΔI (μA)=1.52+

0.854C (nM) and regression coefficient of 0.9988). The de-tection limit was calculated to be 0.078 pM (based on a signal-to-noise ratio of 3, and the noise was determined as thestandard deviation of 3 DPVmeasurements of the blank buffersolution). Notably, this is ~2–4 orders lower than that typicalfor DA aptasensors [25–27], and comfortably meets the re-quirements of even the lowest clinically relevant levels of DAin biological samples [36]. As shown in Table 1, sensingperformances of the voltammetric aptasensor for dopaminebased on PEDOT/rGO are compared with that of other report-ed electrochemical sensor and biosensor systems. Clearly, ourDA aptasensor exhibits both wide linear range and very lowdetection limit.

Specificity of the aptasensor

As noted above, reliable DA quantification is commonlyterminally affected by the presence of interferents. In manybiological fluids, AA, UA and glucose coexist with DA at,normally, substantially higher concentrations. In order to testfor interfacial selectivity herein, the prepared aptasensor re-sponses were assessed in solutions containing DA (50 nM),AA (25.0 μM), UA (25.0 μM) or glucose (25.0 μM) eitherseparately or combined. As shown in Fig. 5, no significantresponse to AA, UA or glucose is observed even when theirpresence exceeds that of DA (compared with the DA re-sponse, their response signal was just 2.02, 1.50 and 1.27 %,respectively). In solutions containing all 4 compounds, theassayed DPV signal differed by only 1.1 % from that associ-ated with a pure DA solution at equivalent concentration.Aptasensor selectivity is, thus, excellent.

Aptasensor regeneration

As noted, one of the valuable features of nucleic acid aptamersis their chemical (and interfacial) stability. In examining the

Fig. 4 DPV measurements of various concentrations of DA in PBS(0.2 M, pH 7.4) with the aptasensor. DA concentrations (curves a-l)were 0.001, 0.003, 0.3, 1.0, 10.0, 30.0, 50.0, 70.0, 90.0, 110.0, 120.0,and 160.0 nM in sequence. Inset is the linear calibration curve of the DAaptasensor

Table 1 Comparison of the electroanalytical performances against other prepared materials for the determination of DA

Analysis methods Material used Analytical range LOD Reference

DPV graphene 4–100 μM 2.64 μM [2]

DPV PEDOT-Ni/Si 12–48 μM 1.5 μM [37]

SWVa AuNPs/PANIoxb 0.15–500 μM 0.03 μM [38]

DPV DNA–PPyoxc 0.3–10 μM 0.08 μM [39]

CVd PPyox/Graphene 25.0–1000 μM 0.1 μM [40]

Amperometric GO/SiO2–MIPse 0.05–160 μM 0.03 μM [41]

CV RNA aptamer/cysteamine 0.1–10 μM 0.1 μM [26]

Amperometric PEDOT/rGO 0.1–175 μM 39 nM [34]

SWV PANI/Graphene/aptamer 0.007–90 nM 1.98 pM [27]

DPV PEDOT/rGO/aptamer 1 pM–160 nM 78 fM Our work

a SWV Squre-wave voltammetry, bPANIox Overoxidized polyaniline, cPPyox Overoxidized polypyrrole, dCV Cyclic voltammetry, eMIPsMolecularlyimprinted polymers

Ultrasensitive dopamine aptasensor

extent to which this support sensor reuse we have been able toshow that regeneration is achievable with high fidelity (seeFig. 6) over 9 generations by immersion in 7.0 M urea for15 min (to disassociate the aptamer-DA complex) and thenPBS washing. It is also clear that the underlying PEDOT/rGOfilm is also stable to repeated use and washing.

Clinical application

The practical application of the prepared aptasensor was eval-uated by assaying DAwithin human serum samples. Sampleswere, specifically, prepared by doping specific concentrationsof DA into human serum samples (obtained from ChengyangPeople’s Hospital in Qingdao, China.), and there was nosample pretreatment (without dilution). The analytical resultsare shown in Table 2 where it is evident that quantification isrobustly retained (assay results being 98.27–100.72 % ofdoped levels), with the relative standard deviation (RSD)ranging from 0.79 to 2.27 %. We believe these observations,which constitute the most effective electroanalytical detectionof DA in any real sample, are the result of excellent aptamer

selectivity in terms of recruitment and high electrocatalyticactivity of the PEDOT/rGO nanocomposite, and the inabilityof any non-specific electroinactive materials to interfere withbound DA coupling to the underlying electrode after thewashing step.

Conclusion

A novel reusable electrochemical label-free aptasensor capa-ble of the reliable detection of DA with ultrahigh sensitivityand selectivity is presented. The readily prepared interfacesare based on the immobilization of aptamer onto an underly-ing conducting polymer PEDOT/rGO of excellent electrocat-alytic activity. The chemically and mechanically robust filmsserve to recruit and preconcentrate DA from solution prior toits direct assessment byDPV. Notably this ability is retained inserum samples and supports a quantification that is accurateacross a broad clinically relevant serum concentration range.We believe the presented results serve as an important exam-ple of the potency associated with marrying specific receptorsto high surface area conductive composite films and can formthe basis for a rapid and convenient quantification ofelectroactive molecules generally in complex biologicalsamples.

Acknowledgments This research was supported by the National Nat-ural Science Foundation of China (No. 21275087, 21175077), the NaturalScience Foundation of Shandong Province of China (ZR2012BM008),and the Taishan Scholar Program of Shandong Province, China.

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Fig. 5 Responses of the aptasensor to 50.0 nMDA (a), 25.0 μMAA (b),25.0 μM UA (c), 25.0 μM glucose (d) and a mixture of 50.0 nM DA,25.0 μM AA, 25.0 μM UA and 25.0 μM glucose. The DPV workingvoltage is ranging from −0.2 to 0.5 V

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Ultrasensitive dopamine aptasensor