An electrochemical aptasensor based on gold nanoparticles and graphene oxide doped poly(3,4-ethylenedioxythiophene) nanocomposite for detection of MUC1

Accepted Manuscript An electrochemical aptasensor based on gold nanoparticles and graphene oxide doped poly(3,4-ethylenedioxythiophene) nanocomposite for detection of MUC1

Pernika Gupta, Anu Bharti, Navpreet Kaur, Suman Singh, Nirmal Prabhakar PII: DOI: Reference:

S1572-6657(18)30098-5 https://doi.org/10.1016/j.jelechem.2018.02.014 JEAC 3866

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

17 November 2017 26 January 2018 6 February 2018

Please cite this article as: Pernika Gupta, Anu Bharti, Navpreet Kaur, Suman Singh, Nirmal Prabhakar , An electrochemical aptasensor based on gold nanoparticles and graphene oxide doped poly(3,4-ethylenedioxythiophene) nanocomposite for detection of MUC1. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jeac(2017), https://doi.org/10.1016/ j.jelechem.2018.02.014

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ACCEPTED MANUSCRIPT An electrochemical aptasensor based on Gold nanoparticles and graphene oxide doped Poly(3,4-ethylenedioxythiophene) nanocomposite for detection of MUC1 Pernika Guptaa#, Anu Bhartia#, Navpreet Kaura, Suman Singhb and Nirmal Prabhakar a* Department of Biochemistry, Panjab University Chandigarh 160014, India

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Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India

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Both have equal contribution

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corresponding author: [email protected]

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Abstract

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*

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In the present work, we have developed an electrochemical aptasensor based on conducting

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polymer nanocomposite for the detection of breast cancer biomarker protein, mucin1 (MUC1). Gold nanoparticles (AuNPs) and graphene oxide (GO) doped Poly(3,4-ethylenedioxythiophene)

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(PEDOT) nanocomposite films were deposited onto the surface of fluorine tin oxide (FTO) glass sheets by electropolymerization using chronoamperometry technique at a potential of 1.0V for

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180s. MUC1 specific biotinylated aptamer was immobilized onto the AuNPs-GO-PEDOT nanocomposite film via biotin-avidin interaction strategy. The biotinylated aptamer immobilized

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electrodes were characterized using contact angle measurement studies, FT-IR and electrochemical techniques. Optimization parameters like aptamer concentration and response

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time were also performed. The developed aptaelectrode was used for the sensing of MUC1 with limit of detection (LOD) of about 1fg/mL (0.031fM). The stability and reusability of the

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aptaelectrodes was observed to be 14 days and 8 times, respectively. The fabricated aptaelectrode was also applied for the determination of MUC1 in spiked human serum samples with 85-93% recovery. Keywords:

Electrochemical;

Aptasensor;

Breast

ethylenedioxythiophene); Gold nanoparticles; Graphene oxide

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cancer;

MUC1;

Poly(3,4

ACCEPTED MANUSCRIPT 1. Introduction Breast cancer is the most common type of malignancy found in women and contributes to about 23% of all cancer cases worldwide [1-3]. Various factors are associated with the onset of cancer which may lead to diverse symptoms on the basis of type and site of tumor development. Advances in breast cancer control are greatly aided by early detection, thereby facilitating

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diagnosis and treatment of breast cancer in its pre-invasive state prior to metastasis. Traditionally available clinical techniques such as mammography, ultrasound, biopsy and MRI

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are routine choice for the detection of about 80-90% of breast cancer cases in women [4]. Apart from these, immunohistochemistry (IHC), enzyme linked immunosorbent assay (ELISA) and

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radioimmunoassay (RIA) are also available for diagnosis of breast cancer. All these techniques are currently in wide use but are associated with false positive or negative results which can

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leads to wrong interpretations and unnecessary biopsies. Therefore, many methods have been emphasized on the development of new minimally invasive, cost effective, quick and point of

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care diagnostic approaches to improve the detection and screening complications. Hence, biosensors are found to be suitable devices for meeting the present diagnostic needs including

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high specificity, sensitivity and response time providing ideal platform for the immobilization of target biomolecule without any external modification [5-8].

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Recently, the discovery of aptamers has shown to provide a number of potential applications in biosensor development due to the sensitive detection of target [9-11]. Aptamers are folded single

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stranded DNA or RNA, consisting about 40-100 nucleotides synthesized artificially through invitro SELEX method [12,13]. Aptamers are considered as an effective alternative over

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antibodies due to its small size, longer shelf life, easier molecular designing and modification, higher reproducibility and stability against denaturation [14,15]. Variation in the expression of surface receptor proteins like HER2, CEA, MUC1, EGFR etc. are considered as indicative for breast cancer progression [16]. These biologically relevant molecules could serve as biomarkers for breast cancer detection. However, the up regulation of MUC1 in breast adenocarcinoma tissues is associated with the ubiquitous and random expression of proteins over the cell surface [17]. These properties and high concentration of MUC1 in blood makes it potentially useful biomarker in breast cancer detection. MUC1 is a high molecular 2

ACCEPTED MANUSCRIPT weight membrane associated glycoprotein which contains a hydrophobic membrane spanning domain, a cytoplasmic domain and an extracellular domain [18,19]. It is found in most human epithelial layers, serves to lubricate and provides protection against mechanical damage and pathological infections [20]. So far, different transducers have been explored for the detection of MUC1 including optical

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[21], mass change [22], surface plasmon resonance [23] and electrochemical [24]. Among all,

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electrochemical biosensors have been considered as preferred choice due to high sensitivity,

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electrochemical kinetics and possibility for miniaturization [25-27]. Very few reports for the detection of MUC1 over expression with its complementary DNA aptamer have been reported using electrochemical biosensing methods. Zhu et al., developed an electrochemical biosensor

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for the detection of MCF-7 cells using MUC1 binding aptamer, for early breast cancer diagnosis [28]. This method is based on aptamer-cell-aptamer sandwich architecture on a gold electrode

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surface with detection limit of 100 cells. Wen et al., reported an Exo-1 assisted electrochemical aptasensor (Exo/MUC1/S2/S1/MCH/Au) for detection of MUC1 with appreciable selectivity and

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sensitivity [29]. Recently, electrochemical detection of MUC-1 with LOD of 24nM has been done using thiolated aptamer immobilized on the surface of AuNPs modified GCE [30]. The

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aptasensor shows good stability and repeatability.

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Numerous immobilization support like carbon based materials (CNTs, graphene, graphene oxide), conducting polymers, metal oxides and their combinations etc. have been used for

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biosensing studies [31]. Poly(3,4-ethylenedioxythiophene) (PEDOT), a widely explored conducting polymer exhibits high electrical conductivity, good electrochemical stability and

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considered as an attractive sensing platform due to its biocompatibility with other molecules [3235]. Graphene oxide has been used to modify the electrode surfaces due to the large surface area, electrical conductivity and excellent thermal as well as chemical stability [36-38]. Activated graphene oxide shows oxygen containing carboxyl groups that provide sites for amide bond formation with the amine groups present in proteins and nucleic acids [39,40]. Gold nanoparticles are being extensively exploited due to their high surface area, small size owing to enhanced catalytic activities [41]. Wang et al., has developed a label free electrochemical aptasensor for the detection of thrombin using graphene based nanocomposite (rGO-AuNPs) [42]. But graphene based ternary composites shows superior performance over binary 3

ACCEPTED MANUSCRIPT composites. Due to the high electrical conductivity, unique environmental stability and superior biocompatibility, conducting polymers has been reported as a promising compound in the field of biosensors [43]. Xue et al., synthesized a gold nanoparticle/polypyrrole/graphene ternary nanocomposite for electrochemical detection of glucose [44]. The biosensor shows an excellent response with high sensitivity signifying the ternary nanocomposite as a promising material for biosensor fabrication. Recently, graphene based ternary nanocomposite (Au-PEDOT/rGO) has

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been used for the detection of caffeic acid (CA) in red wine samples. The LOD of sensor was

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0.004 μM with stability of about 14 day [45]. There is no report available for detection of MUC1

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using this ternary nanocomposite. Therefore, the electrochemical detection of MUC1 using AuNPs-GO-PEDOT nanocomposite based aptasensor has been reported in the present study.

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2. Materials and Methods

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2.1 Chemicals and instrumentation

MUC1 protein, MUC1 specific biotinylated DNA aptamer, 3,4-ethylenedioxythioene (E-DOT), (SA),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

(EDC),

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streptavidin

Hydroxysuccinimide (NHS), Gold (III) chloride hydrate (HAuCl4.3H2O) and fluorine doped tin

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oxide (FTO) sheets were purchased from Sigma-Aldrich (India). The biotinylated aptamer sequence used in the study was 5’-[btn]GGG AGA CAA GAA TAA ACG CTC AAG CAG

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TTG ATC CTT TGG ATA CCC TGG TTC GAC AGG AGG CTC ACA ACA GGC-3’ [23]. Lithium perchlorate (LiOCl4), trisodium citrate, copper sulphate (CuSO4), ethylene diamine tetra

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acetic acid (EDTA), tris hydroxymethyl aminomethane (Tris), ferrous chloride tetrahydrate and ferric chloride hexahydrate were received from Himedia Laboratory Pvt. Ltd., Mumbai, India.

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Sodium dihydrogen orthophosphate (NaH2PO4.2H2O) and di-sodium hydrogen orthophosphate anhydrous (Na2HPO4) were obtained from Thermo Fisher Scientific Pvt. Ltd., Mumbai, India. All the chemicals and reagents used during the experimentation were of analytical grade and used as received without further purification. Aqueous solutions were prepared by using deionized water (dH2O). Phosphate buffer solution (PBS) (50mM, pH 7.0) was prepared by mixing Na2HPO4 (1.42g) and NaH2PO4.2H2O (1.56g) in 200mL of dH2O. TE buffer was prepared by mixing 1mL of tris HCl (10mM, pH 7.5) and 200µL of EDTA (1mM, pH 8.0) and make up total volume 100mL with dH2O.

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ACCEPTED MANUSCRIPT The synthesized AuNPs and GO were characterized by UV-VIS spectroscopy [Shimadzu UV1800]. The prepared electrodes (PEDOT, GO-PEDOT and AuNPs-GO-PEDOT), SA coated AuNPs-GO-PEDOT and aptaelectrode (APT/SA/AuNPs-GO-PEDOT) were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) [Nicolet 1S50 FT-IR] and contact angle studies by Sessile drop method [KRÜSS Drop shape analysis system, Model DSA100]. Electrochemical measurements were performed with Autolab Potentiostat/Galvanostat [AutoLab

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M101, Eco Chemie] composed of three electrode system, an Ag/AgCl as the reference electrode,

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platinum as a auxillary electrode and fabricated FTO surface as working electrode. All the

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studies were conducted in 50mM PBS (pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3-/4solution.

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2.2 Preparation of gold nanoparticles (AuNPs)

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AuNPs were prepared according to the Turkevich citrate method with slight modifications [46,47]. 10mL aqueous solution of 2.5mM HAuCl4 was mixed with 1mL of 0.1mM copper (II)

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sulphate solution and total volume was made 100mL by adding dH2O. The mixture was heated under constant stirring conditions followed by the rapid addition of 5mL 1% (w/v) trisodium

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citrate solution. Color variation was observed subsequently from blue to pink, cherry red and purplish red at the end of the reaction.

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2.3. Synthesis of Graphene oxide (GO)

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GO was synthesized by previously reported method [48]. Graphite powder (2g) was dissolved in a solution of H2SO4:HNO3 (3:1) and refluxed at 40ºC for 16 hours. The resulting suspension was

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filtered. Later on, the filtered GO was dispersed in dH2O and centrifuged five times at 2000rpm for 2 min. to remove all the insoluble particles. Obtained GO was oven dried at 60ºC. For activation, GO powder (20mg) was dissolved in mixed solution of EDC (5mg/mL) and NHS (5mg/mL), kept for 3 hours at room temperature. Finally, the solution was centrifuged at 3000rpm for 15 min. and dried in oven for further use. 2.4. Preparation of aptaelectrode AuNPs-GO-PEDOT films were electropolymerized in a three electrode electrochemical cell using potentiostat. Before modification, the FTO electrode surface was pre-cleaned with 1% 5

ACCEPTED MANUSCRIPT acetic acid, acetone: ethanol (1:1) and finally with dH2O. PEDOT solution was prepared by mixing monomer solution of EDOT (0.5M) and LiOCl4 (0.266g) in acetonitrile: dH2O (2:3). Further, the PEDOT solution containing 1mg/mL GO and 2.5mM AuNPs was subjected to electropolymerization using chronoamperometry at a potential of 1.0V for 180s. Afterwards, 5µL of streptavidin solution (0.05mg/mL) was casted over the surface of GO-AuNPs suspended PEDOT films (AuNPs-GO-PEDOT/FTO) and stored overnight at 4ºC. For immobilization of

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aptamer, 5µL of aptamer solution (15µM) prepared in TE buffer was immobilized on the surface

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of SA modified AuNPs-GO-PEDOT/FTO electrode and incubated overnight at 4ºC. The

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aptaelectrode (APT/SA/AuNPs-GO-PEDOT/FTO) was stored at 4ºC when not in use. Electrodes were washed with dH2O after each step of fabrication for the removal of unbound content.

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Fig. 1 shows the schematic of aptasensor fabrication. The EDOT monomers and GO nanosheets were non-covalently attached through pi-pi stacking interactions during the process of oxidative

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polymerization on FTO electrode. The AuNPs incorporated on the polymerized surface by in situ reduction using sodium citrate as a reducing as well as stabilizing agent. The AuNPs-GO-

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PEDOT modified FTO electrode was incubated with SA to form strong covalent bonds between activated –COOH group of GO and –NH2 groups of SA using EDC-NHS chemistry. The surface

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of SA coated AuNPs-GO-PEDOT/FTO bioelectrode was further modified by incubating biotinylated aptamer specific for MUC1 protein by means of streptavidin-biotin strong bonding.

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The fabricated aptaelectrode (APT/SA/AuNPs-GO-PEDOT/FTO) was characterized to confirm

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the successful immobilization.

2.5. Characterization and optimization studies

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The synthesis of gold nanoparticles and graphene oxide was confirmed using UV-VIS spectroscopy. The contact angle measurement studies and FT-IR characterizations were done to ensure the successful fabrication of aptaelectrode. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed to analyze the changes in current response of various electrodes and bioelectrodes. After successful characterization of aptaelectrode, optimization studies relevant to various parameters such as concentration of aptamer and response time were performed. The different concentrations of aptamer (1µM, 5µM, 10µM, 15µM and 20µM) were used for immobilization 6

ACCEPTED MANUSCRIPT on the surface of SA/AuNPs-GO-PEDOT bioelectrode and kept at 4ºC for 12 hours (Fig. S4). These aptaelectrodes were used for the DPV studies to obtain optimum aptamer concentration required for immobilization. The response time was further optimized by incubating the aptasensor with MUC1 solution prepared in PBS for 5, 10, 15 and 20 min. at room temperature followed by DPV measurements (Fig. S5). The optimized aptamer concentration and response

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time were selected as 15µM and 15 min., respectively.

Fig. 1. Schematic representation for the development of APT/SA/AuNPs-GO-PEDOT coated FTO electrode for breast cancer detection. 2.6. Detection studies The aptaelectrode was incubated for 15 min. with varying concentrations of MUC1 ranging from 0.1fg/mL to 1µg/mL. The electrode was washed in PBS and response was taken in 50mM PBS (pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3-/4- solution.

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ACCEPTED MANUSCRIPT 2.7. Spike in studies The aptaelectrode was also used for the analysis of real samples containing MUC1. Serum samples were spiked with different concentrations of MUC1 (1fg/mL, 1pg/mL, 1ng/mL and 0.1µg/mL) and electrochemically measured using DPV.

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2.8. Regeneration, reusability, storage stability and specificity studies

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To check the performance of aptaelectrode, regeneration, reusability and stability parameters were also investigated. The aptaelectrode can be regenerated very easily by incubating it with

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50mM NaOH solution, for the dissociation of target molecule from electrode surface and washed carefully with dH2O. Now, the electrode can be successively used for further immobilization of

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MUC1 and can be regenerated again for further detection.

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To investigate the storage stability, the developed aptaelectrode was stored at 4ºC for one month. The DPV response was measured after every 7 days for one month. Further the specificity of

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developed aptaelectrode was assessed by DPV response studies of three different target analytes incubated onto the APT/SA/AuNPs-GO-PEDOT surface for 20 min. The aptaelectrode was

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incubated with 1µg/mL of each of Mycobacterium tuberculosis (MPT64), acetylcholinesterase (AChE), bovine serum albumin (BSA) and MUC1 solution prepared in PBS.

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3. Results and discussions

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3.1 Characterization studies 3.1.1.UV-VIS Spectra

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Gold nanoparticles showed an absorption peak at 530nm (Fig. S1A), due to their distinctive feature referred to as localized surface Plasmon resonance (LSPR). The formation of gold nanoparticles with size 20nm was confirmed through its LSPR spectrum as it is dependent on their size and shape [49]. In UV-VIS spectra of GO (Fig. S1B), the absorption peak at 230nm was assigned to p-p* transition of C=C bonds and a shoulder at 290nm corresponds to n-p* transition of C=O bonds [50]. 3.1.1. Contact Angle measurements

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ACCEPTED MANUSCRIPT Contact angle (CA) measurements were carried out to confirm the immobilization of streptavidin and aptamer onto the surface of prepared electrode. The value of contact angle for PEDOT/FTO surface was found to be 38º and decreased to 20º after the addition of GO and AuNPs due to increase in hydrophilicity. In the next step of surface modification with streptavidin, the CA increased to 40º due to fall in hydrophilicity. Slight decrease in CA to 25º after immobilization of aptamer was noted due to increase in hydrophilicity. The constant change in CA results at each

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step of aptaelectrode development confirms the successful fabrication of APT/SA/AuNPs-GO-

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PEDOT (Fig. S2). 3.1.2. FTIR studies

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Fig. 2 shows the FTIR spectra of the prepared bioelectrodes and aptaelectrode. The FTIR spectra of PEDOT [curve (A)] show absorption bands at 1621cm-1, 1340cm-1 and 1042cm-1. The

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absorption band at around 1621cm-1 originates from the inter-ring C=C stretching mode in the quinoidal structure of PEDOT [51]. The C-C stretch of the thiophene ring shows the absorption band at1340cm-1. The band appearing at around 1042cm-1 is attributed to the C-O-C bending in

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ethylenedioxy group while the bands at 865cm-1 and 680cm-1 is due to stretching vibrations of C-

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S-C bond in the thiophene ring [52]. In case of GO-PEDOT [curve (B)], the broad and intense peak at 3361cm-1 originated from the stretching and deformation vibration of O-H bond of GO.

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The COOH group situated at the edges of GO shows a strong band at 1710cm-1 corresponds to the C=O bond. The peaks at 1637cm-1 and 1010cm-1 attributes to the stretching vibrations of

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C=C and C-O (in epoxy group), respectively [53]. After the doping of gold nanoparticles [curve (C)], there is no characteristic peak found, except at 1042cm-1 which corresponds to the C-O-C

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bond of PEDOT. The FTIR spectrum suggest the uniform distribution of AuNPs-GO-PEDOT nanocomposite and presence of all functional groups which are present in GO and PEDOT. The spectra for SA/AuNPs-GO-PEDOT [curve (D)] shows absorption bands at 1650cm-1 (amide-I) and 1490cm-1 (amide-II) indicating successful binding of SA [54]. After the immobilization of aptamer on the surface of SA/AuNPs-GO-PEDOT, the peaks were observed at 1646, 1374, 1244 and 1044cm-1 [curve (E)]. The thymine base present in ssDNA shows the in plane vibrational peak at 1646cm-1 whereas the signals at 1374cm-1 as well as 1044cm-1 corresponds to the C=C and C=N stretching of pyrimidine and purine bases. The phosphate groups present in aptamer sequence shows antisymmetric stretching vibrations at 1244cm-1 [55]. Hence, the results obtained 9

ACCEPTED MANUSCRIPT from the analysis of FTIR spectra indicate successful fabrication and immobilization of prepared

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aptaelectrodes.

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Fig. 2. FT-IR spectra of (A) PEDOT (B) GO-PEDOT (C) AuNP-GO-PEDOT (D) SA/ AuNP-

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GO-PEDOT and (E) APT/SA/AuNP-GO-PEDOT. 3.1.3. Electrochemical characterization CV and EIS studies

Electrochemical characterization of PEDOT/FTO, GO-PEDOT/FTO, AuNPs-GO-PEDOT, SA/AuNPs-GO-PEDOT and aptamer coated SA/AuNPs-GO-PEDOT bioelectrodes was carried out by cyclic voltammetry, as shown in fig. 3(A). The CV studies of the AuNPs-GOPEDOT/FTO, AuNPs-GO/FTO and GO/FTO electrodes as control experiment has been shown in Fig. S3. The results reveal the significant role of PEDOT in improving electrochemical 10

ACCEPTED MANUSCRIPT properties of matrix. The studies were performed in 50mM PBS (pH-7.0, 0.9% NaCl) containing 5mM [Fe(CN)6]3-/4- at scan rate 50mV/s. The CV response of PEDOT (curve i) shows a current response of 0.8mA that increased to 1.15mA after the addition of GO (curve ii). There is further rise in the current upto 2.14mA after incorporation of AuNPs to GO-PEDOT (curve iii). The enhancement in the electrocatalytic behavior after the addition of GOES provide better electrochemical surface area and improves the mechanical strength of PEDOT/GO composite

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whereas gold nanoparticles enhanced the electron transfer efficiency of nanocomposite. The peak

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current experiences an obvious fall after the immobilization of streptavidin (curve iv) as

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streptavidin being protein shows weak conductivity resulting in slow electron transfer and hence low peak current (1.45mA). After aptamer binding the electron transfer efficiency gets improved,

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this might be attributed to the presence of negatively charged phosphate carriers present on aptamer (ssDNA) resulting in enhanced electron transport ability of the bioelectrode showing

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peak current of 1.85mA (curve v). The MUC1 treated aptaelectrode shows a decrease in the peak current (1.58mA) as compared to the APT/SA/AuNPs-GO-PEDOT due to the formation of

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aptamer-target 3D-complex which hinders the diffusion of electrons between [Fe(CN)6]3-/4electrolyte and electrode surface (curve vi).

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To support the result obtained from the CV, the fabricated electrode surfaces were characterized by means of EIS studies shown in fig. 3(B). The nyquist plot represents the charge transfer

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resistance at the electrode or electrolyte interface with the diameter of the semicircle. The R ct values were determined from the diameter of the semicircle in a high-frequency region of the

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nyquist plot. The large diameter of the semicircle corresponded to the material of electrode with a large interfacial resistance and poor charge propagation behavior. The various parameters

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related to EIS data have been presented in Table S1. There is an obvious decrease in the Rct value of GO-PEDOT/FTO (68.4Ω) (curve ii) and AuNPs-GO-PEDOT/FTO (28.7Ω) (curve iii) in comparison to PEDOT/FTO (89.65Ω) (curve i). The AuNPs-GO-PEDOT/FTO nanocomposite exhibited lowest Rct value means less hindrance to the flow of electrons and more conductivity. Immobilization of SA on AuNPs-GO-PEDOT/FTO electrode surface enhanced the Rct value upto 58.7Ω (curve iv) whereas after immobilizing aptamer onto the surface of SA/AuNPs-GOPEDOT/FTO, the electron transfer resistance decreases to 37.9Ω (curve v). The MUC1 treated aptaelectrode shows a slight increase in its Rct value to 55.5Ω (curve vi). Hence, the results are consistent with the CV measurements. As Rct value decreases, resistance to the electron transfer 11

ACCEPTED MANUSCRIPT between [Fe(CN)6]3-/4- and modified electrode also decreases, thus facilitating the increase in

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electrochemical conductivity of prepared electrode.

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Fig. 3. (A) CV responses and (B) EIS spectra of (i) PEDOT (ii) GO-PEDOT (iii) AuNPs-GO-

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PEDOT (iv) SA/AuNPs-GO-PEDOT (v) APT/SA/AuNPs-GO- PEDOT and (vi) MUC1 treated APT/SA/AuNPs-GO- PEDOT in 50mM PBS (pH-7.0, 0.9% NaCl) containing 5mM [Fe(CN)6]3at scan rate 50mV/s.

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Electroactive surface area

The electroactive surface area of various electrodes was determined by recording CV in 50mM

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PBS (pH-7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3-/4- at different scan rates (v) according to Randles–Sevcik equation [56,57]: Ipa = 2.69 × 105 An 3/2CoDr 1/2 v 1/2

equation 1

Where, Ipa = anodic peak current A = surface area of the electrode (cm2) n = number of electrons participating in the redox reaction (1), Dr = diffusion coefficient (7.6 × 10−6 cm2 s−1) 13

ACCEPTED MANUSCRIPT C0 = concentration of [Fe(CN)6]3-/4- (5 mole cm-3) v = scan rate (Vs-1) From the above equation, the electroactive surface area of different electrodes- PEDOT, GOPEDOT, AuNPs-GO-PEDOT, SA/AuNPs-GO-PEDOT and APT/SA/AuNPs-GO-PEDOT were 1.37cm2, 1.4cm2, 1.86cm2, 1.52cm2 and 1.66cm2, respectively. The results confirm that the AuNPs-GO-PEDOT exhibited highest electroactive surface area, this concludes that addition of

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AuNPs and GO into PEDOT contributed toward the improvement of electrochemical properties

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of PEDOT. Hence, AuNPs-GO-PEDOT provides very sensitive platform for immobilization of

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aptaelectrode in electrochemical sensing.

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3.2. Electrochemical detection of MUC1

The aptaelectrodes were used to estimate the MUC1 concentration by DPV curves at a potential

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range of 0.1 – 0.5V. Fig. 4 shows the DPV response of aptaelectrodes with respect to of MUC1 concentration range 0.1fg/mL (3.13aM) -1µg/mL (31.25nM) within 15 min. The results show a

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strong concentration dependent response as the amount of MUC1 protein increases, the active sites for the transfer of electrons decreases resulting in decreased current response. The peak

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current of the electrode with MUC1 protein at concentration 0.1fg/mL is almost equivalent to the bare aptaelectrodes i.e. no aptamer - protein interaction. Hence the limit of detection was

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estimated to be 1fg/mL. Table 1 shows the comparison of the present work with other reported

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literature.

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Fig. 4. DPV response of aptaelectrode incubated with different concentrations of MUC1 (i)

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0fg/mL (ii) 0.1fg/mL (iii) 1fg/mL (iv) 0.01pg/mL (v) 0.1pg/mL (vi) 1pg/mL (vii) 0.01ng/mL (viii) 0.1ng/mL (ix) 0.01µg/mL and (x) 1µg/mL in 50mM PBS (pH-7.0, 0.9% NaCl) containing

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5mM [Fe(CN)6]3-/4- at scan rate 50mV/s.

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Table 1

MUC1. Electrode

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Comparison of APT/SA/AuNPs-GO-PEDOT/FTO based biosensor with others for detection of

Three component DNA system with aQD b anti-CEA-CdS NPs/ c MCF-7/MUC1 aptamer/ gold electrode d Apt-HRP/MUC1/Apt1/Au electrode e LSAW aptasensor based piezoelectric biosensor

Biotransducer

Linear range

Optical

0-2µM

Electrochemical

104-107 cell/mL

3.3×102 cell /mL

Electrochemical

102 -107 cells/mL

100cells/mL 3hours

[28]

Mass change based

1 x 102 -1 x 107 cells/mL

32cells/mL

[22]

15

Detection limit 250nM

Response time 1hour 30min. 2hours

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Ref. [21] [24]

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8.8-353.3nM

2.2nM

20min.

[58]

10 pM-1μM

4pM

60min.

[29]

50-1000nM

24nM

-

[30]

MUC1/APT/SA/AuNPsGO-PEDOT/FTO

3.13aM-31.25nM

0.031fM

15min.

Present study

MCF-7- Michigan cancer foundation-7

d e

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anti-CEA-CdS NPs- Carcinoembryonic antigen antibody-CdS nanoparticles

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b c

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QD- Quantum dot

Apt-HRP- Aptamer-Horse radish peroxidase

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a

Electrochemical

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HO–AuNPs–HRP Electrochemical conjugates/MUC1/strepta vidin/ Chitosang MWCNT-GCE h Exo1/MUCElectrochemical 1/Apt/Capture probe/ i MCH/Au electrode j MB anti-MUC1-aptamer Electrochemical modified GCE/AuNPs

LSAW- Leaky surface acoustic wave

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HO- Hairpin oligonucleotide

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Exo1- Exonuclease-1

MCH- Mercapto-1-hexanol

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MB- Methylene blue

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3.3. Spike in studies

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MWCNT-GCE- Multiwalled carbon nanotubes- glassy carbon electrode

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The APT/SA/AuNPs-GO-PEDOT coated FTO electrode was also tested for its applicability in serum samples. Recovery percentage of aptaelectrodes incubated with different concentration of MUC1 in human serum samples are given in table. 2. The recoveries ranged from 85% to 93.6% for a concentration range of 1fg/mL (3.12 x 10-8nM) to 0.1µg/mL (3.12nM), indicated that the biosensor could be used for MUC1 sensing in real clinical samples. The decrease in recovery % may be due to the non-specific interactions of aptamer with other proteins in serum that make aptamer molecules less available for interaction with its specific target MUC 1 to produce electrochemical response.

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Table 2 Recovery studies of the real samples. Added (nM)

Found (nM)

Recovery (%)

1.

3.12 x 10-8

2.75 x 10-8

88

2.

0.000031

0.000027

3.

0.03125

0.02906

4.

3.12

2.66

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S.No.

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85

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3.4. Regeneration, reusability, storage stability and specificity

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Fig. 5(A) shows the reusability curve of the regenerated aptaelectrode after 8 successive regenerations. The prepared aptaelectrode could retain 88% of its initial value after 8 assays,

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indicating good reproducibility.

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In fig. 5(B), a gradual decrease in the current response from 0 day to 28 days was observed. After 14 days, a 14% drop in the current response was observed and gradually decreased upto

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46% and 86% after 21 and 28 days, respectively. This suggested that the aptaelectrode has relatively good stability for about half month.

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It can be seen in fig. 5(C), that the aptaelectrode shows a significant decrease in the current response with the addition of MUC1 while there is no obvious change in current signals for

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MPT64, AChE and BSA. The results demonstrated that the developed aptaelectrode is highly specific to its target protein, MUC1.

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Fig. 5. (A) Reusability studies of aptaelectrode treated with MUC1 after regeneration with NaOH. (B) Current response of the stored aptaelectrode at the interval of every 7 days. (C)

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Specificity studies of aptaelectrode with MPT64, AChE, BSA and MUC1.

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4. Conclusions

AuNPs and GO significantly improved electrochemical sensing of MUC1 by enhancing the

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electroactive surface area of AuNPs-GO-PEDOT electrode. Thus, AuNPs-GO-PEDOT based aptasensor showed appreciable LOD of 1fg/mL (0.031fM) of MUC1 within 15min. The

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aptasensor could also be reused and found stable for about half a month. The spike in studies offers the potential application of developed aptasensor for early diagnosis of breast cancer by detection of MUC1 serum levels. The performance factor along with sensitivity can further be improved using other nanomaterials like novel metal oxides and some 3D materials. Acknowledgements The authors are thankful to Promotion of University Research and Scientific Excellence Programme of Department of Science and Technology (DST-Purse II) and Special Assistance Programme (UGC-SAP) (F.4-7/2015/DRS-III (SAP-II)) are highly acknowledged. Financial 19

ACCEPTED MANUSCRIPT support received from HRDG (CSIR) for the award of JRFship is also acknowledged. We greatly appreciate the support from Dr. Aman Bhalla, Department of Chemistry, Panjab University, Chandigarh for helping us in performing FT-IR studies. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, M.J. Thun, Cancer Statistics, 2009, CA Cancer.

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Figure captions

FTO electrode for breast cancer detection.

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Fig. 1. Schematic representation for the development of APT/SA/ AuNPs-GO-PEDOT coated

Fig. 2. FT-IR spectra of (A) PEDOT (B) GO-PEDOT (C) AuNP-GO-PEDOT (D) SA/ AuNP-

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GO-PEDOT and (E) APT/SA/AuNP-GO-PEDOT.

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Fig. 3. (A) CV responses and (B) EIS spectra of (i) PEDOT (ii) GO-PEDOT (iii) AuNPs-GOPEDOT (iv) SA/AuNPs-GO- PEDOT (v) APT/SA/ AuNPs-GO- PEDOT and (vi) MUC1 treated /4-

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APT/SA/ AuNPs-GO- PEDOT in 50mM PBS (pH-7.0, 0.9% NaCl) containing 5mM [Fe(CN)6]3at scan rate 50mV/s.

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Fig. 4. DPV response of aptaelectrode incubated with different concentrations of MUC1 (i) 0fg/mL (ii) 0.1fg/mL (iii) 1fg/mL (iv) 0.01pg/mL (v) 0.1pg/mL (vi) 1pg/mL (vii) 0.01ng/mL

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(viii) 0.1ng/mL (ix) 0.01µg/mL and (x) 1µg/mL in 50mM PBS (pH-7.0, 0.9% NaCl) containing

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5mM [Fe(CN)6]3-/4- at scan rate 50mV/s. Fig. 5. (A) Reusability studies of aptaelectrode treated with MUC1 after regeneration with NaOH. (B) Current response of the stored aptaelectrode at the interval of every 7 days. (C) Specificity studies of aptaelectrode with MPT64, AChE, BSA and MUC1.

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Gold nanoparticles (AuNPs) and graphene oxide (GO) doped PEDOT (AuNPs-GO-PEDOT) nanocomposite solution was used for the fabrication of an electrochemical aptasensor to detect MUC1 with LOD 1fg/mL (0.031fM).

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Highlights:AuNPs-GO-PEDOT nanocomposite was used for to develop electrochemical aptasensor. The aptaelectrode shows LOD of 1fg/mL (0.031fM) in response time of 15min. The aptasensor could be reused for 8 times and shows stability for half month. The spike-in sample recovery using serum sample was 85-93%.

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