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Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen
Author’s Accepted Manuscript Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen Esmaeil Heydari-Bafrooei, Shamszadeh
Nazgol
Sadat www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)31297-0 http://dx.doi.org/10.1016/j.bios.2016.12.048 BIOS9441
To appear in: Biosensors and Bioelectronic Received date: 15 September 2016 Revised date: 19 December 2016 Accepted date: 20 December 2016 Cite this article as: Esmaeil Heydari-Bafrooei and Nazgol Sadat Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.12.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen Esmaeil Heydari-Bafrooeia,b*, Nazgol Sadat Shamszadeha a
Department of Chemistry, Faculty of Science, Vali-e-Asr University of Rafsanjan, 77188– 97111, Iran
b
High Temperature Fuel Cell Research Group, Vali-e-Asr University of Rafsanjan, 77188– 97111, Iran E–mail: [email protected]; [email protected] *
Correspondence address: Tel.: +983431312433; Fax: +983431312429.
Abstract A densely packed gold nanoparticles on the rGO-MWCNT platform was used as the basis for an ultrasensitive label-free electrochemical aptasensor to detect the biomarker prostate specific antigen (PSA) in serum. The detection was based on that the variation of electron transfer resistance (Rct) and differential pulse voltammetry (DPV) current were relevant to the formation of PSA–aptamer complex at the modified electrode surface. Compared with pure AuNPs, rGO-MWCNT and MWCNT/AuNPs, the rGO-MWCNT/AuNPs nanocomposite modified electrode was the most sensitive aptasensing platform for the determination of PSA. Two calibration curves were prepared from the data obtained from the DPV and electrochemical impedance spectroscopy (EIS) by plotting the peak current and Rct against PSA concentration, respectively. The proposed aptasensor had an extremely low LOD of 1.0 pg mL-1 PSA within the detection range of 0.005–20 ng mL-1 and 0.005–100 ng mL-1 for DPV and EIS calibration curves, respectively. This sensor exhibited outstanding anti1
interference ability towards co-existing molecules with good stability, sensitivity, and reproducibility. Clinical application was performed with analysis of the PSA levels in serum samples obtained from patients with prostate cancer using both the aptasensor and Immunoradiometric assay. The results revealed the proposed system to be a promising candidate for clinical analysis of PSA. Keywords:
Aptamer;
Prostate
specific
antigen;
Electrochemical
impedance
spectroscopy; Label free detection; Multiwalled carbon naotubes 1. Introduction Cancer is a major global health concern, with approximately 14 million new cases and 8 million cancer-related deaths in 2012, affecting populations in all countries and all regions (Stewart and Wild, 2014). In 2015, there will be an estimated 1,658,370 new cancer cases diagnosed and 589,430 cancer deaths in the US according to the report of American Cancer Society. Cancer is the second most public cause of death in the US, exceeded only by heart disease, and accounts for nearly 1 of every 4 deaths. Among men, lung cancer had the highest incidence (34.2 per 100,000) and prostate cancer had the second highest incidence (31.1 per 100,000) (Stewart and Wild, 2014). However, the treatment of cancer is more effective, if it could be diagnosed at an early stage. Serum prostate-specific antigen (PSA) levels is clinically used as an indicator of the prostate cancer screening, monitoring the efficiency of treatment and evaluating probability of remission post treatment (Partin et al., 1997; Dhanasekaran et al., 2001; Heidenreich et al., 2014). It is a 33-34 kDa single chain glycoprotein with 261 amino acid residues, is produced by both the normal and the diseased prostate cells, but the levels of which are elevated in men with prostate cancer (Heidenreich et al., 2014). Patients with PSA above the cutoff value of 4 ng mL-1 in serum sample are suspected to have prostate cancer (Catalona et al., 1991). Therefore, development of fast and
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low-cost analytical methods for accurate PSA detection is of great importance in the early cancer diagnosis, monitoring the effectiveness of treatment and assessing likelihood of remission post treatment. In recent years, a range of assays have been proposed for PSA detection including electrochemical (Strzeminska et al., 2016; Kavosi et al., 2014; Fan et al., 2016), chemiluminescence (Liu et al., 2016; Liu et al., 2013), electrochemiluminescence (Ma et al., 2016a, 2016b), fluorescence (Liu et al., 2013; Kaya et al., 2015), field effect transistor (Huang et al., 2013; Kim et al., 2009), surface plasmon resonance (Uludag and Tothill, 2012), enzyme-linked immunosorbent assays (Liang et al., 2015), and mass spectrometry (Chen et al., 2015). These assays are mostly based on the use of antibodies as recognition elements. Despite the advantages and extensively use of expensive antibodies in analysis of a variety of proteins, the modification and the in-vivo preparation of antibodies are difficult, high-cost and time consuming (Jolly et al., 2015). Therefore, immunoassays bear high cost and laborious experiments. One of the alternatives to antibodies is aptamers which can offer several advantages over the former. Oligonucleotide aptamers are short and stable singlestranded DNA or RNA sequences that can bind specifically to the target molecule (e.g., drugs, proteins, and other organic or inorganic molecules) with high specificity and affinity, that can be equivalent to those of antibodies as molecular recognition elements in biosensing application (Wang et al., 2015; Heydari-Bafrooei et al., 2016). A DNA aptamer against PSA was recognized by Savory and co-workers in 2010 (Savory et al., 2010) and several methods have also been proposed using aptamer-based PSA biosensing, including optical (Mei et al., 2015; Chen et al., 2012) or electrochemical techniques (Souada et al., 2015; Rahi et al., 2016; Jolly et al., 2016; Yang et al., 2015; Liu et al., 2012; Jolly et al., 2015). Among the various reported aptamer-based biosensors, electrochemical sensors have always been in the spotlight for research due to many merits such as low cost, easy operation,
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high sensitivity, stable and simple instrumentation, rapid response time and portability. To improve the limit of detection (LOD) of analytes by electrochemical sensors, composites of nanomaterials and conducting polymers have also been used (Ensafi et al., 2015; Ensafi et al., 2014a, 2014b, 2014c; Sun et al., 2014). Recently, graphene-based composite materials have triggered extreme importance for scientists due to the synergistic contribution of two or more functional components and many applications (Sun et al., 2014; Gong et al., 2010). Graphene is a kind of well-known nanomaterial with unique properties of high specific surface area, great mechanical strength, high electron mobility under ambient conditions and promising catalytic properties with low manufacturing cost (Qin et al., 2016). In addition, gold nanoparticles (AuNPs) are much used for constructing aptamer-based biosensors due to their ability to increase electronic signals when a biological component is kept in contact with the nanostructured surface (Hsu et al., 2016). AuNPs, with a diameter of 1-100 nm, have a large surface area, superior mechanical, large thermal conducuctivity and a high surface energy to allow ultraeffcient adsorption of proteins with retaining their bioactivity (Sun et al., 2014). Many investigations have demonstrated that graphene-gold nanoparticle nanocomposites have remarkable electrochemical properties (Thanh et al., 2016; Singh et al., 2016; Lian et al., 2014). This nanohybrid can improve the electrochemical response and effective surface area of the electrode. Among the numerous categories of nanostructures, carbon nanotubes (CNTs) hold also a number of exciting potentials in terms of their electron transfer mechanism (Wang et al., 2005) and surface binding phenomenon (Lin et al., 2004). Moreover, multi-walled carbon nanotubes (MWCNT) and AuNPs (MWCNT–AuNPs) composites have been usually used to modify electrodes and the improved effect of the current response is significant (Hou et al., 2016; Li et al., 2016). CNTs have the excellent inplane conductivity, high surface-to-volume ratios and specific surface areas for efficient
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immobilization of a large amount of DNA aptamers with retaining their bioactivity (HeydariBafrooei et al., 2016). In this work, considering benefits of the graphene, AuNPs, and MWCNT nanomaterials, we used them into an aptasensor to apply the synergy contributions on the enhancement of aptasensor characteristics. A 32 bases long DNA aptamer for PSA (Savory et al., 2010) has been used for as a case study and differential pulse voltammetryv (DPV) and electrochemical impedance spectroscopy (EIS) used as a electrochemical technique to monitor PSA levels on aptasensors. Clinical application was performed with analysis of the PSA levels in serum samples obtained from patients with prostate cancer, using both the aptasensor and Immunoradiometric assay. 2. Experimental 2.1. Materials Thiol terminated PSA binding DNA aptamer (5′-HS–(CH2)6 –TTT TTA ATT AAA GCT CGC CAT CAA ATA GCT TT-3′) was purchased from Takapu Zist Institute (Tehran, Iran). PSA was obtained from Merck Chemicals Ltd., UK. All other reagents were of analytical grade and obtained from Sigma-Aldrich, UK. All solutions were prepared by redistilled water. The Aptamer stock solutions were prepared in phosphate buffer solution (PBS) pH 7.0, and kept frozen. The electrolyte solution (K3[Fe(CN)6]/K4[Fe(CN)6] mixture) was immediately prepared before use. 2.2. Synthesis of rGO-MWCNT and immobilization of AuNPs The MWCNT were functionalized to carboxylated MWCNT (MWCNT-COOH) based on earlier reports (Heydari-Bafrooei et al., 2016). Briefly, accurate amounts of weighed asreceived MWCNT were treated with concentrated nitric acid under reflux condition (80 °C, 20 h) with vigorous mixing. The resulting mixture was diluted with water and filtered. Then 5
the solid was washed up to neutral pH, and dried under vacuum at 40 °C overnight. GO was also firstly synthesized from natural graphite powder by a modified Hummers method (see experimental details in the ESI). For the preparation of rGO-MWCNT, 10 mg MWCNT and 10 mL of GO (1 mg mL-1) were mixed and ultrasonicated for 1 h. Finally, the composite was collected and redispersed in D. I. water (1 mg mL-1) after being washed with D. I. water and ethanol for three times and dried. Glassy carbon electrode (GCE, 4.0 mm diameter, Metrohm) was first polished with Al2O3 powder (Aldrich, 0.1, 0.05 mm) using Metrohm polishing kit, and rinsed with deionized water, followed by sonication in ethanol and deionized water. Finally, the GCE were dried under a continuous nitrogen stream at the room temperature. 2.5 μL GO-MWCNT was directly drop cast on the cleaned GCE and allowed to dry. The GOMWCNT modified electrode was then subjected to a constant potential of −0.9 V (vs. Ag/AgCl) for 1000 s in a PBS (0.1 mol L-1, pH 4.2) aqueous solution for the electrochemical reduction of GO to rGO. The electrochemical deposition of AuNPs were prepared by applying a constant potential of -0.2 V in an electrochemical cell containing 1.0 mmol L−1 HAuCl4 solution containing 0.1 mol L−1 KNO3 as electrolyte for 180 s. The as-prepared rGOMWCNT/AuNPs films were rinsed with water and dried. 2.3. Aptamer immobilization onto rGO-MWCNT/AuNPs In order to immobilize the aptamer on the surface of electrode, the modified GCE electrode with rGO-MWCNT/AuNPs was immersed in 0.1 mol L-1 PBS containing aptamer (200 nmol L-1) for an immersion time of 9 h and then the film was rinsed with PBS (0.1 mol L-1, pH 7.0). The aptamer modified electrode was further treated with 1 mmo L-1 6-mercapto1-hexanol for 30 min to obtain a well aligned aptamer monolayer. Then the modified electrode was washed with PBS (0.1 mol L-1, pH 7.0) and double distilled water to remove excess aptamer molecules. 2.4. PSA detection 6
The fabricated electrode was immersed in 0.1 mol L-1 PBS containing PSA with different concentrations for 40 min. Then, the aptasensor was rinsed with PBS (0.1 mol L-1, pH 7.0) to remove non-bonded PSA. The amount of changes in the electrochemical properties of the nanocomposite electrode were detected using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). 2.5. Electrochemical measurements Electrochemical performances of the modified electrodes were conducted by CV, DPV and EIS in a three electrode cell assembly containing 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and 0.1 mol L-1 KCl in PBS (pH 7.0). The EIS was performed from 5 mHz to 100 kHz with AC amplitude set in 10 mV and at a DC potential of +0.15 V versus Ag/AgCl (sat. KCl). The experimental spectra, presented as Nyquist plots, were fitted with proper equivalent circuits using the software supported by the instrument. CV was performed at a scan rate of 100 mV s−1 by potential scanning between -0.2 and 0.5 V. Operating conditions for DPV studies were: step potential of 8.0 mV; modulation amplitude of 50.0 mV; modulation time of 0.05 s and interval time of 0.5 s. All the electroanalytical measurements were performed at room temperature. EIS, DPV, and CV measurements were performed using Autolab PGSTAT302N with NOVA 1.11 software (Eco Chemie, The Netherlands). The electrochemical experimental data of the PSA detection were collected after 3 h at least until the system was stable. 2.7. Characterizations Transmission electron microscopic (TEM) and images were acquired using. The surface characterization was achieved by FE-SEM and TEM using a NOVA NANOSEM 230 and Philips XLC, respectively, at an accelerating voltage of 5 kV and a working distance of 5 mm. Data processing and parametric characterization of the AuNPs, including particles
7
counting (density estimation) and average diameter measurement was performed using MATLAB image processing toolbox software. The density of AuNPs were estimated from a 10.2 µm2 GC surface analysis (counting a minimum of 420 to 891 particles depending on the number of cyclic scans N). For each deposit, the error was calculated from the analysis of three different SEM images. 2.8. Sample preparation Fresh Blood specimens of healthy and patient individuals were gifted by the Alzahra Hospital, Isfahan, Iran, and store frozen until assay. The serum samples were separated and divided into two parts: first recognition by standard immunoradiometric assay and second recognition by the aptasensor. For the determination of PSA using the biosensor these samples were diluted with distilled water (1:9). 3. Results and Discussion Chemical oxidation of MWCNT is recognized to result in the formation of oxygencontaining surface groups including hydroxyl, carboxyl, and carbonyl which act as acids or bases that show ion-exchange properties and improve the dispersibility. GO is also strongly oxygenated, bears functional groups on the hydroxyl and epoxy basal planes, in addition to carbonyl groups and carboxyl groups located on the edges of the floors. The presence of these functional groups makes rGO and MWCNT strongly hydrophilic and therefore readily dispersible in water thus facilitating the drop casting procedures used for the modification of electrode surfaces. After the casting of GO-MWCNT on the surface of the electrode, it is necessary to reduce the GO into rGO. The GO is electrically insulating (Layek and Nandy, 2013) because of the disruption of the sp2 bonding network in its carbon basal plane whereby a significant fraction of the sp2 carbon network is bonded with oxygen-containing functional groups during
8
chemical exfoliation of the graphite. Thus, the graphene oxide has to be reduced to restore the unique properties found in the pristine graphene. X-ray photoelectron spectroscopy (XPS) was used to investigate the efficiency of electrochemical reduction. Core C1s XPS spectra (Figure S-1) showed a dramatic decrease of the C-O bond for electrochemically reduced GO (trace B) when compared to graphene oxide precursor (trace A), likely related to the reduction of epoxides on graphene oxide surfaces. 3.1. Morphology The GO sheets shown in Figure 1A (SEM) and D (TEM) were thin, with a typical flakelike shape and wrinkled texture. Morphologies and microstructures of the original MWCNT were also investigated through SEM and TEM (Figure 1B and E). The CNTs dispersed in water showed a homogeneous configuration and good dispersion. Figure 1C and F exhibit the morphology of MWCNT-GO which CNTs were successfully introduced into graphene or filled between the GO sheets and formed porous hybrid nanostructure. The nanocomposite also displayed a homogeneous configuration, relatively dense and uniform network nanostructure and good dispersion. The synthesized nanohybrid is useful to avoid the aggregation and restacking of GO sheets. Moreover, the MWCNT in the nanocomposites set up a properly conductive network, which may facile charge-transfer and mass-transfer processes. SEM was also used to explore the effect of the electrodeposition time on the morphology of the deposited gold nanostructures. Figure 2 (A-C) shows the SEM photographs of AuNPs deposited onto rGO-MWCNT/GCE substrate by applying constant potential of -0.2 V for (A) 120 (B) 180 and (C) 240 s and its AuNPs size distribution diagram is shown in Figure 2 (DF). It is clear that the figures revel that for the electrodeposition technique, the time of the applied potential is an important factor controlling the morphologies of the gold nanostructures. Increasing the deposition time from 120 to 240 s, the number and size of 9
AuNPs are apparent increased as shown in Figure 2 (D-F). When the electrodeposition time are 120, 180 and 240 s, the average diameter of the Au nanopartices can reach to 38.1, 65.2 and 83.5 nm, respectively. When the deposition time was 120 s, the electrode surface has a smaller size particles, but with lower density (54 ± 3 particles µm-2). On the other hand, when the deposition time was increased to 240 s (Figure 2C), serious aggregation of the gold nanoparticles happened and NPs density reaches 69 ± 4 particles µm-2. AuNPs deposited for 180 s gave uniform, flat and dense crystal grains with clear crystal edges and high density (92 ± 4 particles µm-2). The results confirmed that 180 s is suitable for gold nanoparticles preparation. It is interesting to investigate the C1s XPS spectrum of rGO after applying potential of 0.2 V to rGO modified GCE (rGO/GCE). As shown in the Figure S-1, the XPS spectrum is similar to that of rGO (before applying potential, trace B) and not to that of GO (trace A). Therefore, the surface oxygen functional groups is not formed on the surface of rGO by applying -0.2 V. 3.2. Comparison of electrochemical properties of nanocomposites In order to probe the electrochemical properties of the fabricated aptasensor, its fabrication processes for each modification step studied by CV, DPV and EIS. Figure 3 (A) exhibits the CVs of 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) in 0.1 M PBS (pH 7.0) on the various electrodes
coated
with
GO-MWCNT,
rGO-MWCNT,
and
rGO-MWCNT/AuNPs
nanocomposites at a scan rate of 100 mV s−1. As expected, CV of K3[Fe(CN)6]/K4[Fe(CN)6] on a bare GCE has a pair well-defined redox waves (trace a). The peak current decreased dramatically after coating with GO-MWCNT nanocomposites (trace b). Despite to high electrical conductivity of MWCNT, GO has many functional groups which will hamper the mobility of electrons from the electrolyte to the electrode surface resulting in low conductance of GO-MWCNT as substantiated by CV studies. After self-assembly of rGO10
MWCNT, the redox peak currents clearly increased, which suggested that the electrochemically active sites of the GCE were increased by the rGO-MWCNT as a result of its excellent electron transfer ability, large surface area, and remaining oxygen-related defects in the rGO film (Shams et al., 2016). After the electrode was modified with rGOMWCNT/AuNPs hybrid, the peak currents was further enhanced than that of rGO-MWCNT, indicating that rGO-MWCNT/AuNPs layer could increase surface area and active sites for electron transfer. The highly conductive AuNPs on the rGO sheets behaved as an electron transfer channel, which further improved the conductivity of the rGO-MWCNT (Shams et al., 2016). Furthermore, the DPV results (Figure 3B) are in good agreement with the cyclic voltammograms obtained under similar conditions. Afterwards, the EIS was further performed to probe the process of electrode modification. The data was fitted well with an equivalent circuit (Figure S-2). This circuit includes the resistive and capacitive elements: Rs is the electrolyte resistance, the constant phase element Q1 is then related to the space charge capacitance at the electrode│electrolyte interface, Rct is related to the charge transfer resistance at the electrode│electrolyte interface and the constant phase element Q2 is the Warburg impedance resulting from the diffusion of ions from the bulk of the electrolyte to the interface. The impedance of the constant phase element is explained as: is the modulus, ω the angular phase and n the phase (
⁄ , where Y0
). The constant phase element
shows inhomogeneities and defect areas of the layer. Y0 is the constant of proportionality containing the diffusion coefficient and other parameters which depend on the characteristics of the electrochemical system, j2=−1, ω=2πf is the angular frequency, and n is an exponent. For n= 1, Q models a capacitance with C= Y0; for n=0, a resistance with R= Y0− 1. A special case is obtained for n=0.5, that is, so called Warburg element. This equivalent circuit gives a good description of the impedance data obtained (χ2=0.000008) and allows to calculate the
11
value of Q1, Rct and Q2. In this experiment, the value of n varies from 0.989 to 0.894 which shows that Q1 can be considered as a capacitance C (Tlili et al., 2008). Rct is the most sensitive parameter that responds to changes on the electrode. The semicircle diameter in the Nyquist plot is equal to the Rct. As shown in Figure 3C, the curve a represents an electrochemical impedance of the bare GCE (Rct=755 Ω). By immobilization of the rGO-MWCNT nanocomposite onto the surface of bare GCE, the value of Rct decrease to a much lower resistance compared to the bare GCE due to the higher electron transfer ability and electronic conductivity (Rct=298 Ω). Moreover, when the composite of rGOMWCNT/AuNPs were deposited on the bare GCE, the resistance for the redox probe further decreased (curve d), suggesting that AuNPs were excellent electric conducting materials. Thus, the charge-transfer process between the modified layer and substrate was accelerated. 3.3. PSA detection For the investigation of the importance of rGO-MWCNT/AuNPs nanohybrid for PSA detection, the AuNPs, MWCNT/AuNPs, and rGO-MWCNT/AuNPs modified GCE were prepared and compared with each other. Figure 4 (A, B and C) shows Nyquist plots of the modified electrodes at different steps for the detection of PSA based on three AuNPs-base nanocomposites, i.e., AuNPs (A), MWCNT/AuNPs (B), and rGO-MWCNT/AuNPs (C). When aptamer were modified on the nanomaterial functionalized electrodes surfaces, the Rct increased in sequence (trace c in Figure 4 A-C), which suggested the aptamer severely reduced the effective area and active sites for electron transfer and prevented the access of electrons to the surface and inhibits interfacial charge-transfer (Wang et al., 2015). Upon selfassembly of PSA on the modified electrodes (trace d in Figure 3 A-C), the Rct increases continuously attributed to the steric hindrance generated by the PSA. However, the DPV results of three cases (Figure 4 D, E and F) are in good agreement with the EIS spectra obtained under similar conditions. 12
To assess the performance of each AuNPs-based nanohybrids for PSA detection, Rct and Ip values are summarized in Figure 4 (G and H). Shown on the Y-axis, ∆R and ΔI are difference between Rct and Ip values after and before the immobilization of a new layer adhisive. ∆R and ΔI was increased with incubation of aptasensors with PSA. In contrast, ∆R and ΔI demonstrated a small increase in the case of AuNPs and MWCNT/AuNPs. But PSA detection using the electrochemical biosensor based on the rGO-MWCNT/AuNPs nanocomposite led to the supreme difference in Rct, ΔR=370 Ω and Ip, ΔI=-12.2. The higher ΔR and ΔI of the proposed aptasensor (aptamer on the rGO-MWCNT/AuNPs modified GCE) is attributed to the prescence of rGO-MWCNT as a modifier and excellent electrocatalytic activities of AuNPs on rGO-MWCNT, which performed effective amplification properties as expected. Firstly, the presence of MWCNT not only noticeably increased the surface area to capture more aptamers on the electrode surface but also accelerated electron transfer. Secondly, rGO with high aspect ratio could increase the amount of AuNPs, thus enhanced the response signals. Consequently, the EIS and DPV results (Figure 4) show a synergistic interaction among the three components of the rGO-MWCNT/AuNPs nanocomposite for the immobilization of aptamer molecules. All these phenomena can increase the adsorption amount of DNA onto the composite electrode modified with the rGO-MWCNT/AuNPs nanocomposite. The significant increase in the ΔR and ΔI responses on the rGOMWCNT/AuNPs modified GCE comparing to other modified electrodes, is of interest for the PSA detection. This offers an attractive prospect and novelty for using such films in protein detection using aptamers. Figure 5 A depicts DPVs of 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) in PBS (pH 7.0) at the rGO-MWCNT/AuNPs/Aptamer modified GCE, after binding with different concentrations of PSA prepared in PBS (0.1 mol L-1, pH 7.0). The surface-confined aptamer undergoes a conformation change upon PSA binding in a manner that double stranded is
13
formed with simultaneous binding to (and covering with) PSA that decrease the charge transfer rate (Rct) between redox probe and electrode surface (Rahi et al., 2016). As can be seen, the peak currents decreased with increases of the PSA concentration. The dependency of the ΔI on the PSA concentration is shown in Figure 5B. The calibration plot is linear in the ranges from 0.005 to 20 ng mL-1 with a detection limit (defined as DL=3Sb/m, where m is the slope of the corresponding calibration curve and Sb is the standard deviation of the blank) (Skoog et al., 1998) of 1.0 pg mL-1 for PSA. Figure 5C shows the EIS spectra of rGOMWCNT/AuNPs/Aptamer modified GCE, after binding with various concentrations of PSA prepared in PBS (0.1 mol L-1, pH 7.0). When a PSA molecule combines with an aptamer, the resulting complex would remain on the electrode surface. Because the complex cannot transfer electrons, the conductivity of the overall electrode decreases. Therefore, the resistance increases with the PSA concentration. Therefore, a linear increase in Rct was observed with increase concentration of PSA (logarithmically transformed) (Figure 5D). This is the second calibration curve for PSA detection, with a larger linear behavior (0.005-100 ng mL-1) than that of DPV calibrations. Using the plot, a detection limit of 1.0 pg mL-1 of PSA was attained. As we illustrated in the introduction, the cutoff value of PSA in serum sample is 4 ng mL-1. So, this assay is a promising candidate for simple, sensitive and cost-saving PSA detection in clinic field. Selectivity of proposed aptasensor was evaluated by measuring the DPV and EIS responses. We introduced five PSA analogues such as bovine serum albumin (BSA), hemoglobin, thrombin, human IgG, and lysozyme mixed with PSA (0.5 ng mL-1) following the same experimental conditions. Results are presented in Figure 5 (E and F). As expected, the change in DPV peak current and Rct of the aptasensor corresponding to the addition of BSA (50.0 ng mL-1), hemoglobin (50.0 ng mL-1), thrombin (50.0 ng mL-1), human IgG(50.0 ng mL-1), and lysozyme (50.0 ng mL-1) were negligible, indicating that the selectivity of the
14
developed aptasensor for PSA was good. To further check the selectivity of this aptasensor, the cross-reactivity of other PSA analogues was investigated. As shown in Figure S-3, PSA (0.5 ng mL-1) showed a much stronger change in DPV peak current and Rct of the aptasensor, while nearly insignificant electrochemical changes were sensed for any of the PSA analogues (50.0 ng mL-1). Because the signals was made by the specific interaction of the aptamer and the target molecule, the results displayed that these aptamer could only recognize PSA and could not combine with the PSA analogues. The reproducibility of the aptasensor was investigated by determining 0.5 ng mL-1 PSA solution with five aptasensors which were fabricated following the identical procedure. The relative standard deviation (RSD) of 4.4% was obtained, which indicated that the aptasensor has good reproduction in detection. The repeatability of the aptasensor was also estimated by analyzing one aptasensor for three replicate detections. The RSD of the assay was 4.7%. This indicated that the aptasensor has good repeatability. Stability is a key factor in its practical application for PSA detection. The rGOMWCNT/AuNPs/Aptamer modified GCE was stored at 4 °C for 30 days under optimal conditions. Then we used them to detect the 0.5 ng mL-1 PSA. The current response value decreased approximately 5–8%, which further illustrated the good stability and life length of the aptasensor. We compared our results with various PSA detection methods published in recent years (Table S-1). Our proposed aptasensor, which employed DPV and EIS methods, achieved optimally displaying a lower detection limit, higher sensitivity, strong specificity, and good selectivity. Based on our knowledge, this aptasensor has lowest detection limit and largest linear range among the previously published methods for PSA detection. 3.4. Application of aptasensor
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Although our aptasensor displayed good selectivity for PSA, it is valuable to analyze the analytical effectiveness of the sensor for complex real samples. The detection of PSA in serum is of considerable interest (Table 1). These samples had previously been used in the diagnosis of prostate cancer. The level of PSA was recognized using proposed aptasensor and standard immunoradiometric assay used to assess the performance of the proposed biosensor. The statistical comparisons of the values obtained by these methods for the detection of PSA were made by student’s t-test and the variance ratio of F-test. The experimental t- and Fvalues (at P=0.05) did not exceed the theoretical ones. 4. Conclusion We have successfully demonstrated a novel electrochemical aptasensor for rapid and ultrasensitive detection of PSA in human serum based on the rGO-MWCNT/AuNPs nanocomposite. The rapidity of this method is derived from the inherent rapidity of the electrochemical transducers, and the high sensitivity is due to the high surface-to-volume ratio and good in-plane conductivity caused by the presence of MWCNT, increase the amount of immobilized AuNPs because of presence of the rGO and improvement of the conductivity between the graphene nanosheets due to introduction of the AuNPs, and excellent electrocatalysis activities of AuNPs on rGO-MWCNT, which made effective signal amplification. Therefore, this biosensor, combining the advantages of an aptamer-based method (instead antibody) and the power of an electrochemical detection technique with signal amplification of nanotechnology, provides an option for screening prostate cancer. The aptitude of accurately detecting of PSA in patients serum samples shows the potential for clinical diagnosis of prostate cancer. This new aptasensor offers advantages of lower limit of detection and linearity of response over a wider concentration range over the ELISA as standard testing of PSA. Besides, although the proposed technique was focused on the determination of PSA, it possessed the potential of wider application for other targets. It is 16
expected that such approach based on aptamer will be an attractive tool for the development of new aptasensors. Acknowledgement The authors gratefully acknowledge the support of this work by the Research Council of Vali-e-Asr University of Rafsanjan (VRU), Iran.
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Sun, X., Li, F., Shen, G., Huang, J., Wang, X., 2014. Analyst 139, 299–308. Thanh, T.D., Balamurugan, J., Lee S.H., Kim, N.H., Lee J.H., 2016. Biosens. Bioelectron. 81, 259–267. Tlili, C., Jaffrezic-Renault, N.J., Martelet, C., Korri-Youssoufi, H., 2008. Mater. Sci. Eng. C 28, 848–854. Uludag, Y., Tothill, I.E., 2012. Anal. Chem. 84, 5898–5904. Wang, J., 2005. Electroanalysis 17, 7–14. Wang, M., Zhai, S., Ye, Z., He, L., Donglai, P., Feng, X., Yang, Y., Fang, S., Zhang, H., Zhang, Z., 2015. Dalton Trans. 44, 6473-6479. Yang, Z., Kasprzyk-Hordern, B., Goggins, S., Frost C.G., Estrela, P., 2015. Analyst 140, 2628–2633.
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Table 1. Comparison of proposed aptasensor and Immunoradiometric assay for the detection of PSA in serum. Obtained valuea
Immunoradiometric assaya
(ng mL-1)
(ng mL-1)
A
23.12 ± 0.90
B
Sample
Fex
Ftab
tex
ttab
23.67 ± 0.51
3.1
6.8
1.7
2.3
17.49 ± 0.73
17.23 ± 0.50
2.1
6.8
1.0
2.3
C
62.20 ± 1.10
63.02 ± 0.98
1.3
6.8
1.2
2.3
D
5.12 ± 0.51
5.18 ± 0.40
1.6
6.8
0.4
2.3
a
Each value is the mean of five replicate experiments.
22
Figure captions Figure 1. FE-SEM (A-C) and TEM images (D-F) of synthesized GO (A and D), treated MWCNT (B and E), and rGO-MWCNT nanocomposite (C and F). Figure 2. FE-SEM images (A-C) and size distribution diagram (D-F) of AuNPs modified rGO-MWCNT/GCE. The AuNPs modified electrodes obtained by electrodepositing at the potential of -200 mV in 1.0 mmol L–1 HAuCl4 for (A and D) 120 s, (B and E) 180 s, and (C and F) 240 s. Figure 3. (A) CV and (B) DPV and (C) EIS curves of (a) bare GCE and electrodes containing
(b)
GO-MWCNT,
(c)
rGO-MWCNT
and
(d)
rGO-MWCNT/AuNPs
nanocomposites in a solution containing 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture and 0.1 mol L-1 KCl in PBS (pH 7.0). CV was performed at a scan rate of 100 mV s−1 by potential scanning between -0.2 and 0.5 V. The EIS was performed from 5 mHz to 100 kHz with AC amplitude set in 10 mV and at a DC potential of +0.15 V versus Ag/AgCl (sat. KCl). Operating conditions for DPV studies were: step potential of 8.0 mV; modulation amplitude of 50.0 mV; modulation time of 0.05 s and interval time of 0.5 s. Figure 4. (A) EIS and (B) DPV curves of bare GCE (a), AuNPs modified GCE (b), immobilized aptamer on the AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). (C) EIS and (D) DPV curves of bare GCE (a), MWCNT/AuNPs modified GCE (b), immobilized aptamer on the MWCNT/AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). (E) EIS and (F) DPV curves of bare GCE (a), rGOMWCNT/AuNPs modified GCE (b), immobilized aptamer on the rGO-MWCNT/AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). Variation in Rct (G) and Ip (H) for each stage in PSA detection was measured using developed biosensors, in which AuNPs, MWCNT/AuNPs, and rGO-MWCNT/AuNPs were used as sensitive layers.
23
DPV
and
EIS
curves
obtained
in
a
solution
containing
1.0
mmol
L-1
K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture and 0.1 mol L-1 KCl in PBS (pH 7.0). The operational conditions are as in Fig. 3. Figure 5. (A) DPV curves and (C) Nyquist plots of aptamer-immobilized rGOMWCNT/AuNPs for PSA detection at different concentrations within 0.005-100 ng mL-1. Linear calibration curve for (B) ΔI and (D) ΔR vs. log (CPSA/ng mL-1), where CPSA is the PSA concentration. (E) ΔI and (F) ΔR of the proposed aptasensor corresponding to 0.5 ng mL-1 PSA (a), mixtures of 0.5 ng mL-1 PSA with 50.0 ng mL-1 BSA (b), hemoglobin (c), thrombin (d), human IgG(e), and lysozyme (f), respectively. Error bars represent standard deviation, n=4.
24
25
26
27
28
Highlights
rGO-MWCNT composite was synthesized.
An ultrasensitive electrochemical aptasensor was developed based on the composite for detecting PSA.
The aptasensor displayed low detection limit of 1.0 pg mL-1 for PSA.
The biosensor was used for PSA determination in blood serum, with recovery rates close to 100%.
29
Nazgol
Sadat www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)31297-0 http://dx.doi.org/10.1016/j.bios.2016.12.048 BIOS9441
To appear in: Biosensors and Bioelectronic Received date: 15 September 2016 Revised date: 19 December 2016 Accepted date: 20 December 2016 Cite this article as: Esmaeil Heydari-Bafrooei and Nazgol Sadat Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.12.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen Esmaeil Heydari-Bafrooeia,b*, Nazgol Sadat Shamszadeha a
Department of Chemistry, Faculty of Science, Vali-e-Asr University of Rafsanjan, 77188– 97111, Iran
b
High Temperature Fuel Cell Research Group, Vali-e-Asr University of Rafsanjan, 77188– 97111, Iran E–mail: [email protected]; [email protected] *
Correspondence address: Tel.: +983431312433; Fax: +983431312429.
Abstract A densely packed gold nanoparticles on the rGO-MWCNT platform was used as the basis for an ultrasensitive label-free electrochemical aptasensor to detect the biomarker prostate specific antigen (PSA) in serum. The detection was based on that the variation of electron transfer resistance (Rct) and differential pulse voltammetry (DPV) current were relevant to the formation of PSA–aptamer complex at the modified electrode surface. Compared with pure AuNPs, rGO-MWCNT and MWCNT/AuNPs, the rGO-MWCNT/AuNPs nanocomposite modified electrode was the most sensitive aptasensing platform for the determination of PSA. Two calibration curves were prepared from the data obtained from the DPV and electrochemical impedance spectroscopy (EIS) by plotting the peak current and Rct against PSA concentration, respectively. The proposed aptasensor had an extremely low LOD of 1.0 pg mL-1 PSA within the detection range of 0.005–20 ng mL-1 and 0.005–100 ng mL-1 for DPV and EIS calibration curves, respectively. This sensor exhibited outstanding anti1
interference ability towards co-existing molecules with good stability, sensitivity, and reproducibility. Clinical application was performed with analysis of the PSA levels in serum samples obtained from patients with prostate cancer using both the aptasensor and Immunoradiometric assay. The results revealed the proposed system to be a promising candidate for clinical analysis of PSA. Keywords:
Aptamer;
Prostate
specific
antigen;
Electrochemical
impedance
spectroscopy; Label free detection; Multiwalled carbon naotubes 1. Introduction Cancer is a major global health concern, with approximately 14 million new cases and 8 million cancer-related deaths in 2012, affecting populations in all countries and all regions (Stewart and Wild, 2014). In 2015, there will be an estimated 1,658,370 new cancer cases diagnosed and 589,430 cancer deaths in the US according to the report of American Cancer Society. Cancer is the second most public cause of death in the US, exceeded only by heart disease, and accounts for nearly 1 of every 4 deaths. Among men, lung cancer had the highest incidence (34.2 per 100,000) and prostate cancer had the second highest incidence (31.1 per 100,000) (Stewart and Wild, 2014). However, the treatment of cancer is more effective, if it could be diagnosed at an early stage. Serum prostate-specific antigen (PSA) levels is clinically used as an indicator of the prostate cancer screening, monitoring the efficiency of treatment and evaluating probability of remission post treatment (Partin et al., 1997; Dhanasekaran et al., 2001; Heidenreich et al., 2014). It is a 33-34 kDa single chain glycoprotein with 261 amino acid residues, is produced by both the normal and the diseased prostate cells, but the levels of which are elevated in men with prostate cancer (Heidenreich et al., 2014). Patients with PSA above the cutoff value of 4 ng mL-1 in serum sample are suspected to have prostate cancer (Catalona et al., 1991). Therefore, development of fast and
2
low-cost analytical methods for accurate PSA detection is of great importance in the early cancer diagnosis, monitoring the effectiveness of treatment and assessing likelihood of remission post treatment. In recent years, a range of assays have been proposed for PSA detection including electrochemical (Strzeminska et al., 2016; Kavosi et al., 2014; Fan et al., 2016), chemiluminescence (Liu et al., 2016; Liu et al., 2013), electrochemiluminescence (Ma et al., 2016a, 2016b), fluorescence (Liu et al., 2013; Kaya et al., 2015), field effect transistor (Huang et al., 2013; Kim et al., 2009), surface plasmon resonance (Uludag and Tothill, 2012), enzyme-linked immunosorbent assays (Liang et al., 2015), and mass spectrometry (Chen et al., 2015). These assays are mostly based on the use of antibodies as recognition elements. Despite the advantages and extensively use of expensive antibodies in analysis of a variety of proteins, the modification and the in-vivo preparation of antibodies are difficult, high-cost and time consuming (Jolly et al., 2015). Therefore, immunoassays bear high cost and laborious experiments. One of the alternatives to antibodies is aptamers which can offer several advantages over the former. Oligonucleotide aptamers are short and stable singlestranded DNA or RNA sequences that can bind specifically to the target molecule (e.g., drugs, proteins, and other organic or inorganic molecules) with high specificity and affinity, that can be equivalent to those of antibodies as molecular recognition elements in biosensing application (Wang et al., 2015; Heydari-Bafrooei et al., 2016). A DNA aptamer against PSA was recognized by Savory and co-workers in 2010 (Savory et al., 2010) and several methods have also been proposed using aptamer-based PSA biosensing, including optical (Mei et al., 2015; Chen et al., 2012) or electrochemical techniques (Souada et al., 2015; Rahi et al., 2016; Jolly et al., 2016; Yang et al., 2015; Liu et al., 2012; Jolly et al., 2015). Among the various reported aptamer-based biosensors, electrochemical sensors have always been in the spotlight for research due to many merits such as low cost, easy operation,
3
high sensitivity, stable and simple instrumentation, rapid response time and portability. To improve the limit of detection (LOD) of analytes by electrochemical sensors, composites of nanomaterials and conducting polymers have also been used (Ensafi et al., 2015; Ensafi et al., 2014a, 2014b, 2014c; Sun et al., 2014). Recently, graphene-based composite materials have triggered extreme importance for scientists due to the synergistic contribution of two or more functional components and many applications (Sun et al., 2014; Gong et al., 2010). Graphene is a kind of well-known nanomaterial with unique properties of high specific surface area, great mechanical strength, high electron mobility under ambient conditions and promising catalytic properties with low manufacturing cost (Qin et al., 2016). In addition, gold nanoparticles (AuNPs) are much used for constructing aptamer-based biosensors due to their ability to increase electronic signals when a biological component is kept in contact with the nanostructured surface (Hsu et al., 2016). AuNPs, with a diameter of 1-100 nm, have a large surface area, superior mechanical, large thermal conducuctivity and a high surface energy to allow ultraeffcient adsorption of proteins with retaining their bioactivity (Sun et al., 2014). Many investigations have demonstrated that graphene-gold nanoparticle nanocomposites have remarkable electrochemical properties (Thanh et al., 2016; Singh et al., 2016; Lian et al., 2014). This nanohybrid can improve the electrochemical response and effective surface area of the electrode. Among the numerous categories of nanostructures, carbon nanotubes (CNTs) hold also a number of exciting potentials in terms of their electron transfer mechanism (Wang et al., 2005) and surface binding phenomenon (Lin et al., 2004). Moreover, multi-walled carbon nanotubes (MWCNT) and AuNPs (MWCNT–AuNPs) composites have been usually used to modify electrodes and the improved effect of the current response is significant (Hou et al., 2016; Li et al., 2016). CNTs have the excellent inplane conductivity, high surface-to-volume ratios and specific surface areas for efficient
4
immobilization of a large amount of DNA aptamers with retaining their bioactivity (HeydariBafrooei et al., 2016). In this work, considering benefits of the graphene, AuNPs, and MWCNT nanomaterials, we used them into an aptasensor to apply the synergy contributions on the enhancement of aptasensor characteristics. A 32 bases long DNA aptamer for PSA (Savory et al., 2010) has been used for as a case study and differential pulse voltammetryv (DPV) and electrochemical impedance spectroscopy (EIS) used as a electrochemical technique to monitor PSA levels on aptasensors. Clinical application was performed with analysis of the PSA levels in serum samples obtained from patients with prostate cancer, using both the aptasensor and Immunoradiometric assay. 2. Experimental 2.1. Materials Thiol terminated PSA binding DNA aptamer (5′-HS–(CH2)6 –TTT TTA ATT AAA GCT CGC CAT CAA ATA GCT TT-3′) was purchased from Takapu Zist Institute (Tehran, Iran). PSA was obtained from Merck Chemicals Ltd., UK. All other reagents were of analytical grade and obtained from Sigma-Aldrich, UK. All solutions were prepared by redistilled water. The Aptamer stock solutions were prepared in phosphate buffer solution (PBS) pH 7.0, and kept frozen. The electrolyte solution (K3[Fe(CN)6]/K4[Fe(CN)6] mixture) was immediately prepared before use. 2.2. Synthesis of rGO-MWCNT and immobilization of AuNPs The MWCNT were functionalized to carboxylated MWCNT (MWCNT-COOH) based on earlier reports (Heydari-Bafrooei et al., 2016). Briefly, accurate amounts of weighed asreceived MWCNT were treated with concentrated nitric acid under reflux condition (80 °C, 20 h) with vigorous mixing. The resulting mixture was diluted with water and filtered. Then 5
the solid was washed up to neutral pH, and dried under vacuum at 40 °C overnight. GO was also firstly synthesized from natural graphite powder by a modified Hummers method (see experimental details in the ESI). For the preparation of rGO-MWCNT, 10 mg MWCNT and 10 mL of GO (1 mg mL-1) were mixed and ultrasonicated for 1 h. Finally, the composite was collected and redispersed in D. I. water (1 mg mL-1) after being washed with D. I. water and ethanol for three times and dried. Glassy carbon electrode (GCE, 4.0 mm diameter, Metrohm) was first polished with Al2O3 powder (Aldrich, 0.1, 0.05 mm) using Metrohm polishing kit, and rinsed with deionized water, followed by sonication in ethanol and deionized water. Finally, the GCE were dried under a continuous nitrogen stream at the room temperature. 2.5 μL GO-MWCNT was directly drop cast on the cleaned GCE and allowed to dry. The GOMWCNT modified electrode was then subjected to a constant potential of −0.9 V (vs. Ag/AgCl) for 1000 s in a PBS (0.1 mol L-1, pH 4.2) aqueous solution for the electrochemical reduction of GO to rGO. The electrochemical deposition of AuNPs were prepared by applying a constant potential of -0.2 V in an electrochemical cell containing 1.0 mmol L−1 HAuCl4 solution containing 0.1 mol L−1 KNO3 as electrolyte for 180 s. The as-prepared rGOMWCNT/AuNPs films were rinsed with water and dried. 2.3. Aptamer immobilization onto rGO-MWCNT/AuNPs In order to immobilize the aptamer on the surface of electrode, the modified GCE electrode with rGO-MWCNT/AuNPs was immersed in 0.1 mol L-1 PBS containing aptamer (200 nmol L-1) for an immersion time of 9 h and then the film was rinsed with PBS (0.1 mol L-1, pH 7.0). The aptamer modified electrode was further treated with 1 mmo L-1 6-mercapto1-hexanol for 30 min to obtain a well aligned aptamer monolayer. Then the modified electrode was washed with PBS (0.1 mol L-1, pH 7.0) and double distilled water to remove excess aptamer molecules. 2.4. PSA detection 6
The fabricated electrode was immersed in 0.1 mol L-1 PBS containing PSA with different concentrations for 40 min. Then, the aptasensor was rinsed with PBS (0.1 mol L-1, pH 7.0) to remove non-bonded PSA. The amount of changes in the electrochemical properties of the nanocomposite electrode were detected using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). 2.5. Electrochemical measurements Electrochemical performances of the modified electrodes were conducted by CV, DPV and EIS in a three electrode cell assembly containing 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe and 0.1 mol L-1 KCl in PBS (pH 7.0). The EIS was performed from 5 mHz to 100 kHz with AC amplitude set in 10 mV and at a DC potential of +0.15 V versus Ag/AgCl (sat. KCl). The experimental spectra, presented as Nyquist plots, were fitted with proper equivalent circuits using the software supported by the instrument. CV was performed at a scan rate of 100 mV s−1 by potential scanning between -0.2 and 0.5 V. Operating conditions for DPV studies were: step potential of 8.0 mV; modulation amplitude of 50.0 mV; modulation time of 0.05 s and interval time of 0.5 s. All the electroanalytical measurements were performed at room temperature. EIS, DPV, and CV measurements were performed using Autolab PGSTAT302N with NOVA 1.11 software (Eco Chemie, The Netherlands). The electrochemical experimental data of the PSA detection were collected after 3 h at least until the system was stable. 2.7. Characterizations Transmission electron microscopic (TEM) and images were acquired using. The surface characterization was achieved by FE-SEM and TEM using a NOVA NANOSEM 230 and Philips XLC, respectively, at an accelerating voltage of 5 kV and a working distance of 5 mm. Data processing and parametric characterization of the AuNPs, including particles
7
counting (density estimation) and average diameter measurement was performed using MATLAB image processing toolbox software. The density of AuNPs were estimated from a 10.2 µm2 GC surface analysis (counting a minimum of 420 to 891 particles depending on the number of cyclic scans N). For each deposit, the error was calculated from the analysis of three different SEM images. 2.8. Sample preparation Fresh Blood specimens of healthy and patient individuals were gifted by the Alzahra Hospital, Isfahan, Iran, and store frozen until assay. The serum samples were separated and divided into two parts: first recognition by standard immunoradiometric assay and second recognition by the aptasensor. For the determination of PSA using the biosensor these samples were diluted with distilled water (1:9). 3. Results and Discussion Chemical oxidation of MWCNT is recognized to result in the formation of oxygencontaining surface groups including hydroxyl, carboxyl, and carbonyl which act as acids or bases that show ion-exchange properties and improve the dispersibility. GO is also strongly oxygenated, bears functional groups on the hydroxyl and epoxy basal planes, in addition to carbonyl groups and carboxyl groups located on the edges of the floors. The presence of these functional groups makes rGO and MWCNT strongly hydrophilic and therefore readily dispersible in water thus facilitating the drop casting procedures used for the modification of electrode surfaces. After the casting of GO-MWCNT on the surface of the electrode, it is necessary to reduce the GO into rGO. The GO is electrically insulating (Layek and Nandy, 2013) because of the disruption of the sp2 bonding network in its carbon basal plane whereby a significant fraction of the sp2 carbon network is bonded with oxygen-containing functional groups during
8
chemical exfoliation of the graphite. Thus, the graphene oxide has to be reduced to restore the unique properties found in the pristine graphene. X-ray photoelectron spectroscopy (XPS) was used to investigate the efficiency of electrochemical reduction. Core C1s XPS spectra (Figure S-1) showed a dramatic decrease of the C-O bond for electrochemically reduced GO (trace B) when compared to graphene oxide precursor (trace A), likely related to the reduction of epoxides on graphene oxide surfaces. 3.1. Morphology The GO sheets shown in Figure 1A (SEM) and D (TEM) were thin, with a typical flakelike shape and wrinkled texture. Morphologies and microstructures of the original MWCNT were also investigated through SEM and TEM (Figure 1B and E). The CNTs dispersed in water showed a homogeneous configuration and good dispersion. Figure 1C and F exhibit the morphology of MWCNT-GO which CNTs were successfully introduced into graphene or filled between the GO sheets and formed porous hybrid nanostructure. The nanocomposite also displayed a homogeneous configuration, relatively dense and uniform network nanostructure and good dispersion. The synthesized nanohybrid is useful to avoid the aggregation and restacking of GO sheets. Moreover, the MWCNT in the nanocomposites set up a properly conductive network, which may facile charge-transfer and mass-transfer processes. SEM was also used to explore the effect of the electrodeposition time on the morphology of the deposited gold nanostructures. Figure 2 (A-C) shows the SEM photographs of AuNPs deposited onto rGO-MWCNT/GCE substrate by applying constant potential of -0.2 V for (A) 120 (B) 180 and (C) 240 s and its AuNPs size distribution diagram is shown in Figure 2 (DF). It is clear that the figures revel that for the electrodeposition technique, the time of the applied potential is an important factor controlling the morphologies of the gold nanostructures. Increasing the deposition time from 120 to 240 s, the number and size of 9
AuNPs are apparent increased as shown in Figure 2 (D-F). When the electrodeposition time are 120, 180 and 240 s, the average diameter of the Au nanopartices can reach to 38.1, 65.2 and 83.5 nm, respectively. When the deposition time was 120 s, the electrode surface has a smaller size particles, but with lower density (54 ± 3 particles µm-2). On the other hand, when the deposition time was increased to 240 s (Figure 2C), serious aggregation of the gold nanoparticles happened and NPs density reaches 69 ± 4 particles µm-2. AuNPs deposited for 180 s gave uniform, flat and dense crystal grains with clear crystal edges and high density (92 ± 4 particles µm-2). The results confirmed that 180 s is suitable for gold nanoparticles preparation. It is interesting to investigate the C1s XPS spectrum of rGO after applying potential of 0.2 V to rGO modified GCE (rGO/GCE). As shown in the Figure S-1, the XPS spectrum is similar to that of rGO (before applying potential, trace B) and not to that of GO (trace A). Therefore, the surface oxygen functional groups is not formed on the surface of rGO by applying -0.2 V. 3.2. Comparison of electrochemical properties of nanocomposites In order to probe the electrochemical properties of the fabricated aptasensor, its fabrication processes for each modification step studied by CV, DPV and EIS. Figure 3 (A) exhibits the CVs of 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) in 0.1 M PBS (pH 7.0) on the various electrodes
coated
with
GO-MWCNT,
rGO-MWCNT,
and
rGO-MWCNT/AuNPs
nanocomposites at a scan rate of 100 mV s−1. As expected, CV of K3[Fe(CN)6]/K4[Fe(CN)6] on a bare GCE has a pair well-defined redox waves (trace a). The peak current decreased dramatically after coating with GO-MWCNT nanocomposites (trace b). Despite to high electrical conductivity of MWCNT, GO has many functional groups which will hamper the mobility of electrons from the electrolyte to the electrode surface resulting in low conductance of GO-MWCNT as substantiated by CV studies. After self-assembly of rGO10
MWCNT, the redox peak currents clearly increased, which suggested that the electrochemically active sites of the GCE were increased by the rGO-MWCNT as a result of its excellent electron transfer ability, large surface area, and remaining oxygen-related defects in the rGO film (Shams et al., 2016). After the electrode was modified with rGOMWCNT/AuNPs hybrid, the peak currents was further enhanced than that of rGO-MWCNT, indicating that rGO-MWCNT/AuNPs layer could increase surface area and active sites for electron transfer. The highly conductive AuNPs on the rGO sheets behaved as an electron transfer channel, which further improved the conductivity of the rGO-MWCNT (Shams et al., 2016). Furthermore, the DPV results (Figure 3B) are in good agreement with the cyclic voltammograms obtained under similar conditions. Afterwards, the EIS was further performed to probe the process of electrode modification. The data was fitted well with an equivalent circuit (Figure S-2). This circuit includes the resistive and capacitive elements: Rs is the electrolyte resistance, the constant phase element Q1 is then related to the space charge capacitance at the electrode│electrolyte interface, Rct is related to the charge transfer resistance at the electrode│electrolyte interface and the constant phase element Q2 is the Warburg impedance resulting from the diffusion of ions from the bulk of the electrolyte to the interface. The impedance of the constant phase element is explained as: is the modulus, ω the angular phase and n the phase (
⁄ , where Y0
). The constant phase element
shows inhomogeneities and defect areas of the layer. Y0 is the constant of proportionality containing the diffusion coefficient and other parameters which depend on the characteristics of the electrochemical system, j2=−1, ω=2πf is the angular frequency, and n is an exponent. For n= 1, Q models a capacitance with C= Y0; for n=0, a resistance with R= Y0− 1. A special case is obtained for n=0.5, that is, so called Warburg element. This equivalent circuit gives a good description of the impedance data obtained (χ2=0.000008) and allows to calculate the
11
value of Q1, Rct and Q2. In this experiment, the value of n varies from 0.989 to 0.894 which shows that Q1 can be considered as a capacitance C (Tlili et al., 2008). Rct is the most sensitive parameter that responds to changes on the electrode. The semicircle diameter in the Nyquist plot is equal to the Rct. As shown in Figure 3C, the curve a represents an electrochemical impedance of the bare GCE (Rct=755 Ω). By immobilization of the rGO-MWCNT nanocomposite onto the surface of bare GCE, the value of Rct decrease to a much lower resistance compared to the bare GCE due to the higher electron transfer ability and electronic conductivity (Rct=298 Ω). Moreover, when the composite of rGOMWCNT/AuNPs were deposited on the bare GCE, the resistance for the redox probe further decreased (curve d), suggesting that AuNPs were excellent electric conducting materials. Thus, the charge-transfer process between the modified layer and substrate was accelerated. 3.3. PSA detection For the investigation of the importance of rGO-MWCNT/AuNPs nanohybrid for PSA detection, the AuNPs, MWCNT/AuNPs, and rGO-MWCNT/AuNPs modified GCE were prepared and compared with each other. Figure 4 (A, B and C) shows Nyquist plots of the modified electrodes at different steps for the detection of PSA based on three AuNPs-base nanocomposites, i.e., AuNPs (A), MWCNT/AuNPs (B), and rGO-MWCNT/AuNPs (C). When aptamer were modified on the nanomaterial functionalized electrodes surfaces, the Rct increased in sequence (trace c in Figure 4 A-C), which suggested the aptamer severely reduced the effective area and active sites for electron transfer and prevented the access of electrons to the surface and inhibits interfacial charge-transfer (Wang et al., 2015). Upon selfassembly of PSA on the modified electrodes (trace d in Figure 3 A-C), the Rct increases continuously attributed to the steric hindrance generated by the PSA. However, the DPV results of three cases (Figure 4 D, E and F) are in good agreement with the EIS spectra obtained under similar conditions. 12
To assess the performance of each AuNPs-based nanohybrids for PSA detection, Rct and Ip values are summarized in Figure 4 (G and H). Shown on the Y-axis, ∆R and ΔI are difference between Rct and Ip values after and before the immobilization of a new layer adhisive. ∆R and ΔI was increased with incubation of aptasensors with PSA. In contrast, ∆R and ΔI demonstrated a small increase in the case of AuNPs and MWCNT/AuNPs. But PSA detection using the electrochemical biosensor based on the rGO-MWCNT/AuNPs nanocomposite led to the supreme difference in Rct, ΔR=370 Ω and Ip, ΔI=-12.2. The higher ΔR and ΔI of the proposed aptasensor (aptamer on the rGO-MWCNT/AuNPs modified GCE) is attributed to the prescence of rGO-MWCNT as a modifier and excellent electrocatalytic activities of AuNPs on rGO-MWCNT, which performed effective amplification properties as expected. Firstly, the presence of MWCNT not only noticeably increased the surface area to capture more aptamers on the electrode surface but also accelerated electron transfer. Secondly, rGO with high aspect ratio could increase the amount of AuNPs, thus enhanced the response signals. Consequently, the EIS and DPV results (Figure 4) show a synergistic interaction among the three components of the rGO-MWCNT/AuNPs nanocomposite for the immobilization of aptamer molecules. All these phenomena can increase the adsorption amount of DNA onto the composite electrode modified with the rGO-MWCNT/AuNPs nanocomposite. The significant increase in the ΔR and ΔI responses on the rGOMWCNT/AuNPs modified GCE comparing to other modified electrodes, is of interest for the PSA detection. This offers an attractive prospect and novelty for using such films in protein detection using aptamers. Figure 5 A depicts DPVs of 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) in PBS (pH 7.0) at the rGO-MWCNT/AuNPs/Aptamer modified GCE, after binding with different concentrations of PSA prepared in PBS (0.1 mol L-1, pH 7.0). The surface-confined aptamer undergoes a conformation change upon PSA binding in a manner that double stranded is
13
formed with simultaneous binding to (and covering with) PSA that decrease the charge transfer rate (Rct) between redox probe and electrode surface (Rahi et al., 2016). As can be seen, the peak currents decreased with increases of the PSA concentration. The dependency of the ΔI on the PSA concentration is shown in Figure 5B. The calibration plot is linear in the ranges from 0.005 to 20 ng mL-1 with a detection limit (defined as DL=3Sb/m, where m is the slope of the corresponding calibration curve and Sb is the standard deviation of the blank) (Skoog et al., 1998) of 1.0 pg mL-1 for PSA. Figure 5C shows the EIS spectra of rGOMWCNT/AuNPs/Aptamer modified GCE, after binding with various concentrations of PSA prepared in PBS (0.1 mol L-1, pH 7.0). When a PSA molecule combines with an aptamer, the resulting complex would remain on the electrode surface. Because the complex cannot transfer electrons, the conductivity of the overall electrode decreases. Therefore, the resistance increases with the PSA concentration. Therefore, a linear increase in Rct was observed with increase concentration of PSA (logarithmically transformed) (Figure 5D). This is the second calibration curve for PSA detection, with a larger linear behavior (0.005-100 ng mL-1) than that of DPV calibrations. Using the plot, a detection limit of 1.0 pg mL-1 of PSA was attained. As we illustrated in the introduction, the cutoff value of PSA in serum sample is 4 ng mL-1. So, this assay is a promising candidate for simple, sensitive and cost-saving PSA detection in clinic field. Selectivity of proposed aptasensor was evaluated by measuring the DPV and EIS responses. We introduced five PSA analogues such as bovine serum albumin (BSA), hemoglobin, thrombin, human IgG, and lysozyme mixed with PSA (0.5 ng mL-1) following the same experimental conditions. Results are presented in Figure 5 (E and F). As expected, the change in DPV peak current and Rct of the aptasensor corresponding to the addition of BSA (50.0 ng mL-1), hemoglobin (50.0 ng mL-1), thrombin (50.0 ng mL-1), human IgG(50.0 ng mL-1), and lysozyme (50.0 ng mL-1) were negligible, indicating that the selectivity of the
14
developed aptasensor for PSA was good. To further check the selectivity of this aptasensor, the cross-reactivity of other PSA analogues was investigated. As shown in Figure S-3, PSA (0.5 ng mL-1) showed a much stronger change in DPV peak current and Rct of the aptasensor, while nearly insignificant electrochemical changes were sensed for any of the PSA analogues (50.0 ng mL-1). Because the signals was made by the specific interaction of the aptamer and the target molecule, the results displayed that these aptamer could only recognize PSA and could not combine with the PSA analogues. The reproducibility of the aptasensor was investigated by determining 0.5 ng mL-1 PSA solution with five aptasensors which were fabricated following the identical procedure. The relative standard deviation (RSD) of 4.4% was obtained, which indicated that the aptasensor has good reproduction in detection. The repeatability of the aptasensor was also estimated by analyzing one aptasensor for three replicate detections. The RSD of the assay was 4.7%. This indicated that the aptasensor has good repeatability. Stability is a key factor in its practical application for PSA detection. The rGOMWCNT/AuNPs/Aptamer modified GCE was stored at 4 °C for 30 days under optimal conditions. Then we used them to detect the 0.5 ng mL-1 PSA. The current response value decreased approximately 5–8%, which further illustrated the good stability and life length of the aptasensor. We compared our results with various PSA detection methods published in recent years (Table S-1). Our proposed aptasensor, which employed DPV and EIS methods, achieved optimally displaying a lower detection limit, higher sensitivity, strong specificity, and good selectivity. Based on our knowledge, this aptasensor has lowest detection limit and largest linear range among the previously published methods for PSA detection. 3.4. Application of aptasensor
15
Although our aptasensor displayed good selectivity for PSA, it is valuable to analyze the analytical effectiveness of the sensor for complex real samples. The detection of PSA in serum is of considerable interest (Table 1). These samples had previously been used in the diagnosis of prostate cancer. The level of PSA was recognized using proposed aptasensor and standard immunoradiometric assay used to assess the performance of the proposed biosensor. The statistical comparisons of the values obtained by these methods for the detection of PSA were made by student’s t-test and the variance ratio of F-test. The experimental t- and Fvalues (at P=0.05) did not exceed the theoretical ones. 4. Conclusion We have successfully demonstrated a novel electrochemical aptasensor for rapid and ultrasensitive detection of PSA in human serum based on the rGO-MWCNT/AuNPs nanocomposite. The rapidity of this method is derived from the inherent rapidity of the electrochemical transducers, and the high sensitivity is due to the high surface-to-volume ratio and good in-plane conductivity caused by the presence of MWCNT, increase the amount of immobilized AuNPs because of presence of the rGO and improvement of the conductivity between the graphene nanosheets due to introduction of the AuNPs, and excellent electrocatalysis activities of AuNPs on rGO-MWCNT, which made effective signal amplification. Therefore, this biosensor, combining the advantages of an aptamer-based method (instead antibody) and the power of an electrochemical detection technique with signal amplification of nanotechnology, provides an option for screening prostate cancer. The aptitude of accurately detecting of PSA in patients serum samples shows the potential for clinical diagnosis of prostate cancer. This new aptasensor offers advantages of lower limit of detection and linearity of response over a wider concentration range over the ELISA as standard testing of PSA. Besides, although the proposed technique was focused on the determination of PSA, it possessed the potential of wider application for other targets. It is 16
expected that such approach based on aptamer will be an attractive tool for the development of new aptasensors. Acknowledgement The authors gratefully acknowledge the support of this work by the Research Council of Vali-e-Asr University of Rafsanjan (VRU), Iran.
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Table 1. Comparison of proposed aptasensor and Immunoradiometric assay for the detection of PSA in serum. Obtained valuea
Immunoradiometric assaya
(ng mL-1)
(ng mL-1)
A
23.12 ± 0.90
B
Sample
Fex
Ftab
tex
ttab
23.67 ± 0.51
3.1
6.8
1.7
2.3
17.49 ± 0.73
17.23 ± 0.50
2.1
6.8
1.0
2.3
C
62.20 ± 1.10
63.02 ± 0.98
1.3
6.8
1.2
2.3
D
5.12 ± 0.51
5.18 ± 0.40
1.6
6.8
0.4
2.3
a
Each value is the mean of five replicate experiments.
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Figure captions Figure 1. FE-SEM (A-C) and TEM images (D-F) of synthesized GO (A and D), treated MWCNT (B and E), and rGO-MWCNT nanocomposite (C and F). Figure 2. FE-SEM images (A-C) and size distribution diagram (D-F) of AuNPs modified rGO-MWCNT/GCE. The AuNPs modified electrodes obtained by electrodepositing at the potential of -200 mV in 1.0 mmol L–1 HAuCl4 for (A and D) 120 s, (B and E) 180 s, and (C and F) 240 s. Figure 3. (A) CV and (B) DPV and (C) EIS curves of (a) bare GCE and electrodes containing
(b)
GO-MWCNT,
(c)
rGO-MWCNT
and
(d)
rGO-MWCNT/AuNPs
nanocomposites in a solution containing 1.0 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture and 0.1 mol L-1 KCl in PBS (pH 7.0). CV was performed at a scan rate of 100 mV s−1 by potential scanning between -0.2 and 0.5 V. The EIS was performed from 5 mHz to 100 kHz with AC amplitude set in 10 mV and at a DC potential of +0.15 V versus Ag/AgCl (sat. KCl). Operating conditions for DPV studies were: step potential of 8.0 mV; modulation amplitude of 50.0 mV; modulation time of 0.05 s and interval time of 0.5 s. Figure 4. (A) EIS and (B) DPV curves of bare GCE (a), AuNPs modified GCE (b), immobilized aptamer on the AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). (C) EIS and (D) DPV curves of bare GCE (a), MWCNT/AuNPs modified GCE (b), immobilized aptamer on the MWCNT/AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). (E) EIS and (F) DPV curves of bare GCE (a), rGOMWCNT/AuNPs modified GCE (b), immobilized aptamer on the rGO-MWCNT/AuNPs modified GCE (c), and aptamer coordinated with PSA (1.0 ng mL- 1) (d). Variation in Rct (G) and Ip (H) for each stage in PSA detection was measured using developed biosensors, in which AuNPs, MWCNT/AuNPs, and rGO-MWCNT/AuNPs were used as sensitive layers.
23
DPV
and
EIS
curves
obtained
in
a
solution
containing
1.0
mmol
L-1
K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture and 0.1 mol L-1 KCl in PBS (pH 7.0). The operational conditions are as in Fig. 3. Figure 5. (A) DPV curves and (C) Nyquist plots of aptamer-immobilized rGOMWCNT/AuNPs for PSA detection at different concentrations within 0.005-100 ng mL-1. Linear calibration curve for (B) ΔI and (D) ΔR vs. log (CPSA/ng mL-1), where CPSA is the PSA concentration. (E) ΔI and (F) ΔR of the proposed aptasensor corresponding to 0.5 ng mL-1 PSA (a), mixtures of 0.5 ng mL-1 PSA with 50.0 ng mL-1 BSA (b), hemoglobin (c), thrombin (d), human IgG(e), and lysozyme (f), respectively. Error bars represent standard deviation, n=4.
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26
27
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Highlights
rGO-MWCNT composite was synthesized.
An ultrasensitive electrochemical aptasensor was developed based on the composite for detecting PSA.
The aptasensor displayed low detection limit of 1.0 pg mL-1 for PSA.
The biosensor was used for PSA determination in blood serum, with recovery rates close to 100%.
29