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Electrochemical aptamer-based bioplatform for ultrasensitive detection of prostate specific antigen
Sensors & Actuators: B. Chemical 297 (2019) 126762
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical aptamer-based bioplatform for ultrasensitive detection of prostate specific antigen Amal Raouafia,b, Alfredo Sánchezb, Noureddine Raouafia, , Reynaldo Villalongab, ⁎
T
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a
Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Faculty of Science, University of Tunis El Manar, 2092, Tunis El Manar, Tunisia b Nanosensors and Nanomachines Group, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040, Madrid, Spain
ARTICLE INFO
ABSTRACT
Keywords: Aptamer PSA Biosensing" Graphene SCPE Competition
A novel and disposable potentio-amperometric aptasensor for the prostate specific antigen (PSA) was constructed using functionalized graphene-modified carbon screen-printed electrodes as transducing surface. The PSA specific DNA aptamer was covalently tethered to the graphene through amide bond between the aptamerterminated amine and the carboxylic acid-enriched graphene casted on the electrode surface. A further hybridization of a partially complementary DNA (cDNA) was followed by intercalation of methylene blue into the double-stranded DNA sequences. The detection approach was based on a competitive assay between the antigen and the cDNA. In fact, the detection relies on the PSA biorecognition by its aptamer, triggering the release of the loosely bound DNA strand and the intercalated dye molecules, which was monitored by differential pulse voltammetry. The aptasensor allowed selective and specific detection of PSA over a wide range of concentrations from 1 pg·mL−1 to 100 ng·mL−1 with a low detection limit of 0.064 pg·mL-1. This electroanalytical device also exhibited high reproducibility and storage stability, and was successfully validated in spiked human blood serum samples.
1. Introduction To improve access of a large population to healthcare services, it is of a paramount importance to provide universal, amenable and lowcost point-of-care tools for the screening and early detection of the most common forms of diseases [1]. In 2013, the World Cancer Declaration set nine targets to be reached by 2020, the sixth one is indeed the “universal access to screening and early detection for cancer” [2]. Furthermore, miniaturization of biosensing devices is useful for use in remote areas and conflict zones lacking electricity and basic infrastructures. Electrochemical biosensor can be operated using low voltage provided by disposable batteries such as in glucometers. Aptamers are single-stranded DNA or RNA oligonucleotides and have attracted great attention in biosensing as specific receptors and in medicine as blockers of biological receptors such as the inhibition of the activity of a protein by blocking its active site or preventing the interaction of a protein with another protein, DNA or receptor [3], and for fast screening of biomarkers diseases [4]. Aptamer-based biosensing devices rely on aptamers to replace antibodies, which are prone to degradation and are usually expensive [5]. Aptamer-based methods present myriad of advantages over the traditional antibody assays in
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early cancer detection. Their main merits lie on high selectivity, affinity and stability thus have the ability to differentiate between isoforms and splice variants of a protein giving them high potential for multiple applications even in multiplex discovery platforms [6]. Furthermore, the stability of aptamers, which can be even regenerated after denaturation, increases the shelf life of a commercial aptamer-based detection kit [7]. Cancers are in the top of malignant disease needing detection in their early stages to increase the overall survival rates. According to the European Association of Urology, prostate cancer is one of the most common cancers dealing with male gender [8]. It had the second highest incidence in male health in USA [9]. In 2012, 417.000 new cases were diagnosed in the European Union. Incidence rates of prostate cancer vary by more than 7-fold (25–193 per 100.000), the highest rates were estimated in Northern and Western European countries and the lowest in Central and Eastern European countries [10]. This cancer is classified the 4th in all types of cancer worldwide and is the most frequent type of cancer for men in 91 countries [11]. The reference biomarker for prostate cancer diagnosis is the prostate specific antigen, which is a specific glycoprotein, secreted in human blood sera. The normal levels of PSA are lower than 4 ng·mL−1. Levels between 4 and
Corresponding authors. E-mail addresses: [email protected] (N. Raouafi), [email protected] (R. Villalonga).
https://doi.org/10.1016/j.snb.2019.126762 Received 7 January 2019; Received in revised form 31 May 2019; Accepted 29 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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10 ng·mL−1 are indicative of serious problems for prostate performance [12,13]. Among these disorders we mention prostate cancer. So far, a lot of work has been devoted to develop new methods to detect PSA. Aptasensing platforms for PSA detection using fluorescence [14,15], photoelectrochemistry [16], surface-enhanced Raman scattering [17], enzyme-linked immunoassay [18] and electrochemical techniques such as DPV and electrochemical impedance spectroscopy [19,20] were recently reported. Associated to their unique physicochemical properties, nanomaterials offer great promise for numerous aptasensing systems. For example, carbon nanomaterials-based printed electrodes are used in different applications such as detection of biomolecules, small molecules, proteins, DNA and nucleic acids in general [21]. Coating detection platforms with nanomaterials improves the sensitivity of biosensors, offers the possibility to anchor a variety of biological receptors on the surface and expands the sensing platform applications. Herein, a disposable electrochemical aptasensor for the ultrasensitive detection of prostate specific antigen, based on a SPCE modified with GO-CO2H and functionalized with a selective anti-PSA aptamer. The rational biosensing strategy relies on the exchange of a complementary DNA sequence hybridized with the bioreceptor by the target glycoprotein. This competitive displacement provokes the release of methylene blue (MB) molecules intercalated in the double-stranded DNA sequence, resulting in a decrease of the electrochemical signal of the redox probe.
drop casting of 5 μL on the surface of working electrode and dried for 24 h at RT. Before immobilization of the aminated aptamer, the carboxylic groups of GO-CO2H/SPCE electrode were activated using an EDC/NHS aqueous solution (1 mg·mL−1) for 30 min. Subsequently, 5 μL of anti-PSA aptamer (50 μM) in the working buffer (0.1 M PB solution, pH 7.4) were dropped onto the electrode surface and were allowed to react during 30 min in humid chamber. The electrode was gently rinsed after each modification to avoid blocking the surface with the excess of reagents. The unbound carboxylic groups were treated with an excess of ethanolamine (5 μL, 20 mM in water) for 30 min. Afterward, the hybridization between the complementary DNA (5 μL, 50 μM in 0.1 M PB solution, pH 7.4) and anti-PSA aptamer was left for 30 min, then the electrode was washed with water. As final step, methylene blue molecules were intercalated between the two strands by incubation with 10 μL of MB solution (5 mM) for 30 min. Finally, the electrode was washed with the working buffer and kept at 4 °C until use. 2.3. Detection of the analyte The different concentrations of PSA were dissolved in sodium phosphate buffer (pH 7.4). All measurements were recorded in PB solution (pH 7.4) using the following parameters: step potential 0.005 V, pulse potential 0.05 V, pulse time 0.025 s and scan rate 0.05 V·s–1. To perform the detection, 5 μL of PSA solution were dropped on the surface of the modified electrode and allowed to react for 30 min to ensure the well recognition between the aptamer and the antigen. Afterward, the electrode was washed thoroughly. The quantification of PSA was measured in current reduction and potential shift values using differential pulse voltammetry.
2. Materials and methods 2.1. Reagents and apparatus Table 1 summarizes the DNA oligonucleotides purchased from Sigma Aldrich (Spain, www.sigmaaldrich.com). PSA was acquired from Merck Millipore (Massachusetts, USA, www. merckmillipore.com). Methylene blue, ethanolamine, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), 1,1-ferrocenedimethanol 97% (Fc(CH2OH)2), human blood sera, human serum albumin (HSA), human immunoglobulin G (IgG) and carcinoembryonic antigen (CEA) were purchased from Sigma Aldrich. Screen-printed carbon electrodes OHT-000 and GO-CO2H were kindly donated by Orion High Technologies (Madrid, Spain, www.orion-hitech.com). All other reagents were of analytical grade. All electrochemical measurements were carried out with an EmStat3 Blue potentiostat (PalmSens, The Netherlands, www.palmsens. com) containing a screen-printed electrode connector. PSTrace 4.8 software was used to collect and fit data. Modified SPCEs were used as transducers consisting of a 4-mm diameter working electrode, a carbon counter electrode and an Ag/AgCl reference electrode. All experiments were run in triplicate at room temperature (RT). Scanning electron microscope (SEM) images were collected using JEOL JSM 7600 F microscope.
3. Results and discussion 3.1. Design of the bioplatform The aptasensor assembly and biosensing approach employed in the electrochemical detection of PSA is depicted in Fig. 1. Initially, the working electrode of a SPCE was coated with the acidfunctionalized reduced graphene oxide. This nanomaterial provides the surface with carboxylic functional groups and the exceptional physicochemical properties of the graphene derivatives [22,23]. The interactions of graphene with various types of biorecognition molecules have provided effective tools to develop efficient high-performance analytical devices [24–26]. First, the carboxylic groups were activated using EDC/NHS chemistry [27] followed, in a second step, by the addition of the amine-terminated aptamer. Thereafter, ethanolamine was added to block the unreacted carboxylic groups. This will help avoiding nonspecific reactions on the sensing surface. After hybridization of cDNA with the aptamer, MB is intercalated between the two strands due to its affinity to guanine [28]. Upon the addition of the target, the aptamer binds specifically with PSA due to favorable thermodynamics and especially since the cDNA is not fully complementary to the sensing sequence. The PSA recognition by the aptamer triggers the release of the cDNA from the sensing surface and consequently the decrease of the methylene blue signal that was intercalated between the two sequences.
2.2. Bioplatform assembly 1 mg of GO-CO2H powder was suspended in 1 mL distilled water. The suspension was sonicated for three hours to obtain a homogeneous solution, stable at RT. The electrode was modified with GO-CO2H by
3.2. Effect of the nanomaterial and optimization of the parameters The effect of the modification of SPCE surface with the GO-CO2H nanomaterial was evaluated by cyclic voltammetry, as displayed in Fig. 2. After coating the electrode by graphene, the cathodic and anodic currents of the redox probe [Fe(CN)6]3/4− decrease, which is due to the electrostatic repulsion between the carboxylate groups onto the electrode surface and the negatively charged redox probe (Fig. 2A). A neutral redox probe, Fc(CH2OH)2, was also examined to study the effect of GO-CO2H on the conductivity of SPCE. Presence of the nanomaterial caused a noticeable increase in the electron-transfer of the
Table 1 DNA oligonucleotides. Anti-PSA aptamer
5´-NH2-(CH2)6 TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3′
Complementary sequence (cDNA)
5´-NH2-(CH2)6 –GCTATTTGATTTTTTTTTTTTTT-3′
2
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Fig. 1. Schematic representation of the biosensor assembly and operation.
Fig. 2. Cyclic voltammograms of bare SPCE (curve a) and GO-CO2H-modified SPCE (curve b) in 5 mM [Fe(CN)6]3/4- (A) and 1 mM Fc(CH2OH)2 (B) in 0.1 M KCl solution at 100 mV·s–1.
ferrocenedimethanol probe on the electrode surface (Fig. 2B), which can be ascribed to the high electric conductivity of the graphene [29]. Therefore, the amounts of the aptamer and the cDNA employed in the biosensor assembly were optimized by cyclic voltammetry using Fc (CH2OH)2 as a redox probe (Fig. 3). Optimization experiments were performed by incubating the activated GO-CO2H and aptamer/GO-CO2H modified electrodes in different concentrations of the aptamer and the cDNA, respectively. We observed that the redox probe current decreases with the increase of concentrations of each oligonucleotide to level off at 50 μM for both as it can be seen in Fig. 3. Thus, the high amount of the aptamer immobilized on the electrode surface is essential to obtain sensitive detection of the target analyte. For further studies, a concentration of 50 μM of the aptamer and cDNA were used.
The stepwise assembly of the aptasensor was characterized by cyclic voltammetry in presence of Fc(CH2OH)2 (Fig. 4). As described above, we noticed that the coating of electrode with GO-CO2H resulted in an increase of the current intensity, even more remarkable in the cathodic peak. Subsequently, the immobilization of the aptamer provoked the blocking of the electrode surface yielding a substantial reduction of the signal. Furthermore, we remarked that the sequential addition of ethanolamine and cDNA induced small changes in the current suggesting the successful blocking of the surface. Similar results were achieved by electrochemical impedance spectroscopy (EIS). The experimental data were fitted by using an equivalent circuit model (inset of Fig. 4B). EIS plot for the SPCE shows an enlarged semicircle with a diameter of RCT equal to 39.86 kΩ. However, after been coated with GO-CO2H layer, the surface modification induces Fig. 3. Cyclic voltammograms recorded with the GO-CO2H/SPCE in 0.1 M KCl solution containing 1 mM Fc(CH2OH)2 (A) before (a) and after immobilization of the aptamer at different concentrations: 0.5 (b), 5 (c), 10 (d), 50 (e) and 100 μM (f). (B) After the hybridization of the cDNA on anti-PSA aptamer/ GO-CO2H/SPCE using different cDNA concentrations: 0 (a), 1 (b), 10 (c), 50 (d) and 100 μM (e).
3
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Fig. 4. (A) CVs and (B) EIS of SPCE recorded in 0.1 M KCl solution containing 1 mM of Fc(CH2OH)2 before (a) and after sequential modifications with GO-CO2H (b), aptamer immobilization (c), ethanolamine addition (d) and hybridization with the complementary DNA (e).
a dramatic decrease of RCT as revealed in the decrease of the semicircle diameter at high frequencies in the corresponding Nyquist plots (0.96 kΩ). The GO-CO2H film greatly improves the conductivity and the electron transfer process. The RCT slowly increases after the stepwise modification by the aptamer (2.16 kΩ) then the cDNA (10.62 kΩ). The elaboration of the biosensor was also visualized by scanning electron microscopy showing changes in the surface for the different steps (Figure 1S).
single-stranded DNA via favorable π―π stacking interactions with its adjacent base pairs [35]. The MB bound to dsDNA makes it easier to be reduced than when it is linked to ssDNA. The use of such hybridization indicator is a great method to discriminate between ssDNA and dsDNA, which result in potential shift and current variation. This biosensor has the advantage to be amperometric and potentiometric at the same time. A relationship linking the differential pulse voltammogram current to the logarithm of the analyte concentration over five decades of concentrations ranging from 1 pg·mL−1 to 100 ng·mL-1 was established. The linear regression equation was:
3.3. Sensing of the analyte
ΔI (μA) = 0.269 log[PSA] (pg·mL–1) + 3.444
First, we examined the use of ferrocenedimethanol, under the optimized conditions, as a redox probe potentially able to evaluate the PSA biorecognition. However, as it is shown in Fig. 2S, at different PSA concentrations the signal remained unchanged, and hence it is not useful as biosensing approach. As second tentative, we tested the biosensor with ferri/ferrocyanide but we encountered the same behavior of ferrocenedimethanol (Fig. 3S), then we opted for a different approach. The intercalation of MB molecules is well recognized in aptasensing since this organic dye is endowed with redox properties and high affinity to dsDNA [28,30–33]. This dye is reducible to its leucomethylene blue after the exchange of two electrons and one proton reaction at physiological pH [34]. As it can be seen in Fig. 5A, the MB current decreased with the increase of PSA concentration in the range from 1 pg·mL−1 to 500 ng mL−1, suggesting the release of MB and the loosely bound DNA oligonucleotide after the addition of analyte. The MB redox signals shifted to more cathodic potentials, which reflect different environment of MB before and after binding of the target analyte to the active surface. To explain this behavior, we suggest that MB is probably linked to the backbone negatively charged phosphate via electrostatic interactions in the case of single-stranded DNA. Although, MB is more prone to bind to double-stranded DNA than to
The correlation coefficient (R2) is 0.999 and the limit of detection is 0.064 pg·mL−1. This parameter was calculated according to the 3Sb/m criterion, where m is the slope of the calibration curve and Sb was estimated as the standard deviation of three different DPV signals recorded for the lowest analyte concentration measured. A logarithmic relationship was also elucidated between the potential values and the analyte concentration in the same linear range according the following equation: ΔE (V) = 0.01 log[PSA] (pg·mL–1) + 0.163 With a limit of detection equal to 1 pg·mL−1 and a correlation coefficient (R²) of 0.991. The comparison of our biosensor performances with previously reported electrochemical biosensors shows the excellent behavior of our system for PSA detection (Table 2). It allows the detection of the antigen over a wide range of concentrations with the lowest limit of detection.
Fig. 5. (A) Differential pulse voltammograms of MB immobilized in the aptasensing system recorded in 0.1 M PB solution (pH 7.4) after sequential additions of different concentrations of PSA ranging from 1 pg·mL−1 to 500 ng mL−1. (B) Calibration plots of the developed aptasensor for PSA detection. 4
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Table 2 Comparison of some reported biosensors with the present work. Detection system
Method
Range (ng·mL−1)
LOD (ng·mL−1)
reference
Au/apta-MIP GCE/poly(JUG-co-JUGA)/Apta electrode Au/MSF/apta GCE/rGO-MWCNT/AuNPs/apta
capacitance SWV DPV DPV EIS DPV DPV
0.1 to 100 1 to 104 1 to 300 0.005 to 20 0.005 to 100 0.25 to 200 0.001- 100
0.001 1 0.28 0.001
[36] [37] [38] [39]
0.25 0.064 × 10−3
[40] Present work
PGE/AuNP@GMC/apta SPCE/GO-CO2H/apta/cDNA/MB
LOD: limit of detection, Au: gold electrode, Apta: anti-PSA aptamer, MIP: molecularly imprinted polymer, GCE: glassy carbon electrode, p(JUG-co-JUGA): poly(5hydroxy-1,4- naphthoquinone-co- 3-(5-hydroxy-1,4-dihydro-1,4-dioxonaphthalen-2(3)yl)propionic acid), SWV: square wave voltammetry, MSF: Mesoporous silica thin films, rGO: reduced graphene oxide, MWCNT: multi-walled carbon nanotubes, AuNPs: gold nanoparticles, PGE: pyrolytic graphite electrode, AuNP@GMC: gold nanoparticles encapsulated in graphitized mesoporous carbons.
3.5. Stability, reproducibility and applicability in complex matrix The aptasensor stability was studied by following the change in the DPV signal of MB for two weeks for a set of electrodes stored at 4 °C. We observed that biosensors retained ca. 98% of their initial currents showing good stability and detection reliability (Fig. 4S). The reproducibility of the biosensor was studied using three batches of electrodes, each batch contain three electrodes, over three periods of time. We tested them with a 100 pg·mL−1 concentration of PSA. The first batch was taken from the calibration curve of the detection, the second after 8 months and the third after 10 months. The results prove that the system is reproducible in time (Fig. 5S). Since our aptasensor exhibited good selectivity for PSA in presence of the major proteins existing in human blood sera, we ought to confirm this selectivity in more complex samples such as blood sera. We used the device to determine PSA in spiked human sera. The detection process was the same as that in spiked buffered solutions. The blood sera were diluted twice in order to decrease their viscosity. The analytical results were gathered in Table 3. The values of the measured concentrations after successive additions of the analyte to the blood serum solution are close to those added ones. Furthermore, the recovery values are between 99% and 104%, which demonstrate that the method can be reasonably applied for physiological liquids.
Fig. 6. Selectivity (Dark cyan) and specificity (Violet) tests in the presence of 200 pg·mL−1 of each interferent (IgG, CEA and HSA), 130 mM of Na+, 4 mM of K+ and 100 pg·mL−1 of PSA. The error bars are calculated from three independent measurements. Table 3 Determination of PSA in human blood sera using the developed aptasensor. Samples
Added (P) (pg·mL−1)
Found (F) (pg·mL−1)
Recoverya (%)
Human blood sera
0 10 100
ND1 9.92 103.8
– 99 ± 6 104 ± 6
a
4. Conclusions We have successfully developed a novel disposable electrochemical biosensor based on GO-CO2H modified SPCE for rapid and sensitive PSA detection in buffered solutions and complex matrices such as human blood sera. The detection strategy is based on a competitive process between PSA recognized by the aptamer and the DNA strand which hybridized to the aptamer. MB, employed as redox-active indicator, is intercalated between the two DNA strands. Recognition of the antigen results in the release of MB molecules and cDNA, thus the decrease of the DPV signal and the shift of potential to cathodic values. The prepared aptasensor performance shows a sensitive detection of PSA with a good detection limit in 5-decades range of concentrations and it is able to quantify the antigen in complex matrix.
Average of three determinations ± standard deviation, ND: not detected.
3.4. Selectivity and specificity To validate the biosensor analytical performances, we challenged it against three different interferents present in human blood sera. It was found that an excellent discrimination was achieved in presence of HSA, CEA and IgG. PSA gave a neat decrease in current however the other three interferents with 2-fold concentration (200 pg·mL−1) do not provoke any significant change in the currents denoting a very good selectivity toward PSA. Na+ and K+ were also tested with their physiological concentrations. They had a little effect on the current intensity compared to the reaction with PSA (Fig. 6). Specificity of the aptasensor was also investigated. The biosensor was tested by measuring and comparing the response of the as-mentioned interferents together in presence and in absence of the target analyte. No noticeable current change was observed from the mixture without PSA compared to the mixture with PSA. The results indicate a good specificity of the developed biosensing system.
Acknowledgments The authors acknowledge the financial support from the Tunisian Ministry of Higher Education and Scientific Research (lab support for LR99ES15), Spanish Ministry of Economy and Competitiveness (MINECO Projects CTQ2014-58989-P and CTQ2015-71936-REDT). AR thanks the University of Tunis El Manar for the “Bourse d’Alternance” mobility grant and ANEC (ISP-funded network) for the financial support. 5
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Appendix A. Supplementary data [23]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126762.
[24]
References
[25]
[1] E.T.S.G. da Silva, D.E.P. Souto, J.T.C. Barragan, J.F. Giarola, A.C.M. de Moraes, L.T. Kubota, Electrochemical biosensors in point‐of‐care devices: recent advances and future trends, ChemElectroChem 4 (2017) 778–794. [2] The Refreshed World Cancer Declaration, (2019) (visited 10/11/2018), https:// www.uicc.org/refreshed-world-cancer-declaration. [3] G. Kaur, I. Roy, Therapeutic applications of aptamers, Expert Opin. Investig. Drugs 17 (2008) 43–60. [4] Y. Zhang, Y. Chen, D. Han, I. Ocsoy, W. Tan, Aptamers selected by cell-SELEX for application in cancer studies, Bioanalysis 2 (2010) 907–918. [5] V. Thiviyanathan, D.G. Gorenstein, Aptamers and the next generation of diagnostic reagent, Proteomics Clin. Appl. 6 (2012) 563–573. [6] J.C. Lai, H.E. Horng, C.Y. Hong, Multiplex immunoassays utilizing diferential afnity using aptamers generated by MARAS, Sci. Rep. 7 (2017) 6397. [7] P.B. Crulhas, A.E. Karpik, F.K. Delella, G.R. Castro, V.A. Pedrosa, Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells, Anal. Bioanal. Chem. 409 (2017) 6771–6780. [8] N. Mottet, J. Bellmunt, M. Bolla, E. Briers, M.G. Cumberbatch, M. De Santis, N. Fossati, T. Gross, A.M. Henry, S. Joniau, T.B. Lam, M.D. Mason, V.B. Matveev, P.C. Moldovan, R.C.N. Van den Bergh, T. Van den Broeck, H.G. Van der Poel, T.H. Van der Kwast, O. Rouviere, I.G. Schots, T. Wiegel, P. Cornford, EAU-ESTROSIOG Guidelines on prostate cancer. Part 1: screening, diagnosis, and local treatment with curative intent, Eur. Urol. 71 (2017) 618–629. [9] E. Heydari-Bafrooeia, N.S. Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosens. Bioelectron. 91 (2017) 284–292. [10] J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, S. Rosso, J.W.W. Coebergh, H. Comber, D. Forman, F. Bray, Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012, Eur. J. Cancer 49 (2013) 1374–1403. [11] F. Bray, L.A. Kiemeney, Epidemiology of prostate cancer in Europe: patterns, trends and determinants, Management of Prostate Cancer, Springer, 2017, pp. 1–27. [12] Y.-L. Deng, D.-D. Xu, D.-W. Pang, H.-W. Tang, Target-triggered signal turn-on detection of prostate specific antigen based on metal enhanced fluorescence of Ag@ SiO2@SiO2- RuBpy composite nanoparticles, Nanotechnology 28 (2016) 501–510. [13] T.T.N. Do, T.V. Phi, T.P. Nguy, P. Wagner, K. Eersels, M.C. Vestergaard, L.T.N. Truong, Anisotropic in situ-coated aunps on screen-printed carbon surface for enhanced prostate-specific antigen impedimetric aptasensor, J. Electron. Mater. 46 (2017) 3542–3552. [14] R.-M. Kong, L. Ding, Z. Wang, J. You, F. Qu, A novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor for sensitive detection of prostate specific antigen, Anal. Bioanal. Chem. 407 (2015) 369–377. [15] B.-Y. Fang, C.-Y. Wang, C. Li, H.-B. Wang, Y.-D. Zhao, Amplified using DNase I and aptamer/graphene oxide for sensing prostate specific antigen in human serum, Sens. Actuators B Chem. 244 (2017) 928–933. [16] Q. Zhou, Y. Lin, J. Shu, K. Zhang, Z. Yu, D. Tang, Reduced graphene oxide-functionalized FeOOH for signal-on photoelectrochemical sensing of prostate-specific antigen with bioresponsive controlled release system, Biosens. Bioelectron. 98 (2017) 15–21. [17] T. Hao, X. Wu, L. Xu, L. Liu, W. Ma, H. Kuan, C. Xu, Ultrasensitive detection of Prostate-Specific Antigen and Thrombin based on gold-upconversion nanoparticle assembled pyramids, Small 13 (2017) 1603944. [18] M. Bielefeld-Sévigny, AlphaLISA Immunoassays: the no-wash alternative to ELISAs for research and drug discovery, Assay Drug Dev. Technol. 7 (2009) 90–92. [19] B. Kavosi, A. Salimi, R. Hallaj, F. Moradi, Ultrasensitive electrochemical immunosensor for PSA biomarker detection in prostate cancer cells using gold nanoparticles/PAMAM dendrimer loaded with enzyme linked aptamer as integrated triple signal amplification strategy, Biosens. Bioelectron. 74 (2015) 915–923. [20] P. Jolly, V. Tamboli, R.L. Harniman, P. Estrela, C.J. Allender, J.L. Bowen, AptamerMIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen, Biosens. Bioelectron. 75 (2016) 188–195. [21] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: a review, Electroanalysis 22 (2010) 1027. [22] J. Ping, Y. Zhou, Y. Wu, V. Papper, S. Boujday, R.S. Marks, T.W.J. Steele, Recent
[26] [27] [28] [29]
[30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
advances in aptasensors based on graphene and graphene-like nanomaterials, Biosens. Bioelectron. 64 (2015) 373–385. M.S. Artiles, C.S. Rout, T.S. Fisher, Graphene-based hybrid materials and devices for biosensing, Adv. Drug Deliv. Rev. 63 (2011) 1352–1360. A. Tabasi, A. Noorbakhsh, E. Sharifi, Reduced graphene oxide-chitosan-aptamer interface as new platform for ultrasensitive detection of human epidermal growth factor receptor 2, Biosens. Bioelectron. 95 (2017) 117–123. Q. Wang, Z. Zhou, Y. Zhai, L. Zhang, W. Hong, Z. Zhang, S. Dong, Label-free aptamer biosensor for thrombin detection based on functionalized graphene nanocomposites, Talanta 141 (2015) 247–252. S. Deng, V. Tjoa, H.M. Fan, H.R. Tan, D.C. Sayle, M. Olivo, S. Mhaisalkar, J. Wei, C.H. Sow, Reduced graphene oxide conjugated Cu2O nanowire mesocrystals for high-performance NO2 gas sensor, J. Am. Chem. Soc. 134 (2012) 4905–4917. D.-H. Jung, B.H. Kim, Y.K. Ko, M.S. Jung, S. Jung, S.Y. Lee, H.-T. Jung, Covalent attachment and hybridization of DNA oligonucleotides on patterned single-walled carbon nanotube films, Langmuir 20 (2004) 8886–8891. K. Kerman, D. Ozkan, P. Kara, B. Meric, J.J. Gooding, M. Ozsoz, Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes, Anal. Chim. Acta 462 (2002) 39–47. K.Y. Goud, A. Hayat, G. Catanante, M. Satyanarayana, K.V. Gobi, J.L. Marty, An electrochemical aptasensor based on functionalized graphene oxide assisted electrocatalytic signal amplification of methylene blue for aflatoxin B1 detection, Electrochim. Acta 244 (2017) 96–103. S. Nafisi, A.A. Saboury, N. Keramat, J.-F. Neault, H.-A. Tajmir-Riahi, Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue, J. Mol. Struct. 827 (2007) 35–43. A. Erdem, K. Kerman, B. Meric, U.S. Akarca, M. Ozsoz, Novel hybridization indicator methylene blue for the electrochemical detection of short DNA sequences related to the hepatitis B virus, Anal. Chim. Acta 422 (2000) 139–149. T. Hianik, J. Wang, Electrochemical aptasensors – recent achievements and perspectives, Electroanalysis 21 (2009) 1223–1235. E. Farjami, L. Clima, K.V. Gothelfa, E.E. Ferapontova, DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific, Analyst 135 (2010) 1443–1448. H. Ju, J. Zhou, C. Cai, H. Chen, The electrochemical behavior of methylene blue at a microcylinder carbon fiber electrode, Electroanalysis 7 (1995) 1165–1170. H.-A. Rafiee-Pour, M. Behpour, M. Keshavarz, A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-2, Biosens. Bioelecron. 77 (2016) 202–207. P. Jolly, V. Tamboli, R.L. Harniman, P. Estrela, C.J. Allender, J.L. Bowen, Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen, Biosens. Bioelecron. 75 (2016) 188–195. M. Souada, B. Piro, S. Reisberg, G. Anquetin, V. Noël, M.C. Pham, Label-free electrochemical detection of prostate specific antigen based on nucleic acid aptamer, Biosens. Bioelecron. 68 (2015) 49–54. W. Argoubi, A. Sanchez, C. Parrado, N. Raouafi, R. Villalonga, Label-free electrochemical aptasensing platform based on mesoporous silica thin film for the detection of prostate specific antigen, Sens. Actuators B Chem. 255 (2018) 309–315. E. Heydari-Bafrooe, N.S. Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosens. Bioelectron. 91 (2017) 284–292. B. Liu, L. Lu, E. Hua, S. Jiang, G. Xie, Detection of the human prostate-specific antigen using an aptasensor with gold nanoparticles encapsulated by graphitized mesoporous carbon, Microchim. Acta 178 (2012) 163–170.
Amal Raouafi is a Ph.D. student in Chemistry working at the University of Tunis El Manar, Tunisia. Her research is focused on the design of electrochemical biosensors based on carbon nanomaterials for biological applications. Alfredo Sanchez is a Ph.D. in Chemistry. He is currently a Postdoc Researcher at Complutense university of Madrid, Spain. Noureddine Raouafi is currently of professor of Chemistry at the University of Tunis El Manar and director of the Analytical Chemistry and Electrochemistry Laboratory in the as mentioned university. His research interests are devoted to the design of electrochemical nano(bio)sensors for health care, environmental monitoring and food safety. Reynaldo Villalonga is a Professor of Analytical Chemsitry and head of the Nanosensors and Nanomachines group at Complutense University of Madrid, Spain.
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical aptamer-based bioplatform for ultrasensitive detection of prostate specific antigen Amal Raouafia,b, Alfredo Sánchezb, Noureddine Raouafia, , Reynaldo Villalongab, ⁎
T
⁎
a
Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Faculty of Science, University of Tunis El Manar, 2092, Tunis El Manar, Tunisia b Nanosensors and Nanomachines Group, Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040, Madrid, Spain
ARTICLE INFO
ABSTRACT
Keywords: Aptamer PSA Biosensing" Graphene SCPE Competition
A novel and disposable potentio-amperometric aptasensor for the prostate specific antigen (PSA) was constructed using functionalized graphene-modified carbon screen-printed electrodes as transducing surface. The PSA specific DNA aptamer was covalently tethered to the graphene through amide bond between the aptamerterminated amine and the carboxylic acid-enriched graphene casted on the electrode surface. A further hybridization of a partially complementary DNA (cDNA) was followed by intercalation of methylene blue into the double-stranded DNA sequences. The detection approach was based on a competitive assay between the antigen and the cDNA. In fact, the detection relies on the PSA biorecognition by its aptamer, triggering the release of the loosely bound DNA strand and the intercalated dye molecules, which was monitored by differential pulse voltammetry. The aptasensor allowed selective and specific detection of PSA over a wide range of concentrations from 1 pg·mL−1 to 100 ng·mL−1 with a low detection limit of 0.064 pg·mL-1. This electroanalytical device also exhibited high reproducibility and storage stability, and was successfully validated in spiked human blood serum samples.
1. Introduction To improve access of a large population to healthcare services, it is of a paramount importance to provide universal, amenable and lowcost point-of-care tools for the screening and early detection of the most common forms of diseases [1]. In 2013, the World Cancer Declaration set nine targets to be reached by 2020, the sixth one is indeed the “universal access to screening and early detection for cancer” [2]. Furthermore, miniaturization of biosensing devices is useful for use in remote areas and conflict zones lacking electricity and basic infrastructures. Electrochemical biosensor can be operated using low voltage provided by disposable batteries such as in glucometers. Aptamers are single-stranded DNA or RNA oligonucleotides and have attracted great attention in biosensing as specific receptors and in medicine as blockers of biological receptors such as the inhibition of the activity of a protein by blocking its active site or preventing the interaction of a protein with another protein, DNA or receptor [3], and for fast screening of biomarkers diseases [4]. Aptamer-based biosensing devices rely on aptamers to replace antibodies, which are prone to degradation and are usually expensive [5]. Aptamer-based methods present myriad of advantages over the traditional antibody assays in
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early cancer detection. Their main merits lie on high selectivity, affinity and stability thus have the ability to differentiate between isoforms and splice variants of a protein giving them high potential for multiple applications even in multiplex discovery platforms [6]. Furthermore, the stability of aptamers, which can be even regenerated after denaturation, increases the shelf life of a commercial aptamer-based detection kit [7]. Cancers are in the top of malignant disease needing detection in their early stages to increase the overall survival rates. According to the European Association of Urology, prostate cancer is one of the most common cancers dealing with male gender [8]. It had the second highest incidence in male health in USA [9]. In 2012, 417.000 new cases were diagnosed in the European Union. Incidence rates of prostate cancer vary by more than 7-fold (25–193 per 100.000), the highest rates were estimated in Northern and Western European countries and the lowest in Central and Eastern European countries [10]. This cancer is classified the 4th in all types of cancer worldwide and is the most frequent type of cancer for men in 91 countries [11]. The reference biomarker for prostate cancer diagnosis is the prostate specific antigen, which is a specific glycoprotein, secreted in human blood sera. The normal levels of PSA are lower than 4 ng·mL−1. Levels between 4 and
Corresponding authors. E-mail addresses: [email protected] (N. Raouafi), [email protected] (R. Villalonga).
https://doi.org/10.1016/j.snb.2019.126762 Received 7 January 2019; Received in revised form 31 May 2019; Accepted 29 June 2019 Available online 02 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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10 ng·mL−1 are indicative of serious problems for prostate performance [12,13]. Among these disorders we mention prostate cancer. So far, a lot of work has been devoted to develop new methods to detect PSA. Aptasensing platforms for PSA detection using fluorescence [14,15], photoelectrochemistry [16], surface-enhanced Raman scattering [17], enzyme-linked immunoassay [18] and electrochemical techniques such as DPV and electrochemical impedance spectroscopy [19,20] were recently reported. Associated to their unique physicochemical properties, nanomaterials offer great promise for numerous aptasensing systems. For example, carbon nanomaterials-based printed electrodes are used in different applications such as detection of biomolecules, small molecules, proteins, DNA and nucleic acids in general [21]. Coating detection platforms with nanomaterials improves the sensitivity of biosensors, offers the possibility to anchor a variety of biological receptors on the surface and expands the sensing platform applications. Herein, a disposable electrochemical aptasensor for the ultrasensitive detection of prostate specific antigen, based on a SPCE modified with GO-CO2H and functionalized with a selective anti-PSA aptamer. The rational biosensing strategy relies on the exchange of a complementary DNA sequence hybridized with the bioreceptor by the target glycoprotein. This competitive displacement provokes the release of methylene blue (MB) molecules intercalated in the double-stranded DNA sequence, resulting in a decrease of the electrochemical signal of the redox probe.
drop casting of 5 μL on the surface of working electrode and dried for 24 h at RT. Before immobilization of the aminated aptamer, the carboxylic groups of GO-CO2H/SPCE electrode were activated using an EDC/NHS aqueous solution (1 mg·mL−1) for 30 min. Subsequently, 5 μL of anti-PSA aptamer (50 μM) in the working buffer (0.1 M PB solution, pH 7.4) were dropped onto the electrode surface and were allowed to react during 30 min in humid chamber. The electrode was gently rinsed after each modification to avoid blocking the surface with the excess of reagents. The unbound carboxylic groups were treated with an excess of ethanolamine (5 μL, 20 mM in water) for 30 min. Afterward, the hybridization between the complementary DNA (5 μL, 50 μM in 0.1 M PB solution, pH 7.4) and anti-PSA aptamer was left for 30 min, then the electrode was washed with water. As final step, methylene blue molecules were intercalated between the two strands by incubation with 10 μL of MB solution (5 mM) for 30 min. Finally, the electrode was washed with the working buffer and kept at 4 °C until use. 2.3. Detection of the analyte The different concentrations of PSA were dissolved in sodium phosphate buffer (pH 7.4). All measurements were recorded in PB solution (pH 7.4) using the following parameters: step potential 0.005 V, pulse potential 0.05 V, pulse time 0.025 s and scan rate 0.05 V·s–1. To perform the detection, 5 μL of PSA solution were dropped on the surface of the modified electrode and allowed to react for 30 min to ensure the well recognition between the aptamer and the antigen. Afterward, the electrode was washed thoroughly. The quantification of PSA was measured in current reduction and potential shift values using differential pulse voltammetry.
2. Materials and methods 2.1. Reagents and apparatus Table 1 summarizes the DNA oligonucleotides purchased from Sigma Aldrich (Spain, www.sigmaaldrich.com). PSA was acquired from Merck Millipore (Massachusetts, USA, www. merckmillipore.com). Methylene blue, ethanolamine, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), 1,1-ferrocenedimethanol 97% (Fc(CH2OH)2), human blood sera, human serum albumin (HSA), human immunoglobulin G (IgG) and carcinoembryonic antigen (CEA) were purchased from Sigma Aldrich. Screen-printed carbon electrodes OHT-000 and GO-CO2H were kindly donated by Orion High Technologies (Madrid, Spain, www.orion-hitech.com). All other reagents were of analytical grade. All electrochemical measurements were carried out with an EmStat3 Blue potentiostat (PalmSens, The Netherlands, www.palmsens. com) containing a screen-printed electrode connector. PSTrace 4.8 software was used to collect and fit data. Modified SPCEs were used as transducers consisting of a 4-mm diameter working electrode, a carbon counter electrode and an Ag/AgCl reference electrode. All experiments were run in triplicate at room temperature (RT). Scanning electron microscope (SEM) images were collected using JEOL JSM 7600 F microscope.
3. Results and discussion 3.1. Design of the bioplatform The aptasensor assembly and biosensing approach employed in the electrochemical detection of PSA is depicted in Fig. 1. Initially, the working electrode of a SPCE was coated with the acidfunctionalized reduced graphene oxide. This nanomaterial provides the surface with carboxylic functional groups and the exceptional physicochemical properties of the graphene derivatives [22,23]. The interactions of graphene with various types of biorecognition molecules have provided effective tools to develop efficient high-performance analytical devices [24–26]. First, the carboxylic groups were activated using EDC/NHS chemistry [27] followed, in a second step, by the addition of the amine-terminated aptamer. Thereafter, ethanolamine was added to block the unreacted carboxylic groups. This will help avoiding nonspecific reactions on the sensing surface. After hybridization of cDNA with the aptamer, MB is intercalated between the two strands due to its affinity to guanine [28]. Upon the addition of the target, the aptamer binds specifically with PSA due to favorable thermodynamics and especially since the cDNA is not fully complementary to the sensing sequence. The PSA recognition by the aptamer triggers the release of the cDNA from the sensing surface and consequently the decrease of the methylene blue signal that was intercalated between the two sequences.
2.2. Bioplatform assembly 1 mg of GO-CO2H powder was suspended in 1 mL distilled water. The suspension was sonicated for three hours to obtain a homogeneous solution, stable at RT. The electrode was modified with GO-CO2H by
3.2. Effect of the nanomaterial and optimization of the parameters The effect of the modification of SPCE surface with the GO-CO2H nanomaterial was evaluated by cyclic voltammetry, as displayed in Fig. 2. After coating the electrode by graphene, the cathodic and anodic currents of the redox probe [Fe(CN)6]3/4− decrease, which is due to the electrostatic repulsion between the carboxylate groups onto the electrode surface and the negatively charged redox probe (Fig. 2A). A neutral redox probe, Fc(CH2OH)2, was also examined to study the effect of GO-CO2H on the conductivity of SPCE. Presence of the nanomaterial caused a noticeable increase in the electron-transfer of the
Table 1 DNA oligonucleotides. Anti-PSA aptamer
5´-NH2-(CH2)6 TTTTTAATTAAAGCTCGCCATCAAATAGCTTT-3′
Complementary sequence (cDNA)
5´-NH2-(CH2)6 –GCTATTTGATTTTTTTTTTTTTT-3′
2
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Fig. 1. Schematic representation of the biosensor assembly and operation.
Fig. 2. Cyclic voltammograms of bare SPCE (curve a) and GO-CO2H-modified SPCE (curve b) in 5 mM [Fe(CN)6]3/4- (A) and 1 mM Fc(CH2OH)2 (B) in 0.1 M KCl solution at 100 mV·s–1.
ferrocenedimethanol probe on the electrode surface (Fig. 2B), which can be ascribed to the high electric conductivity of the graphene [29]. Therefore, the amounts of the aptamer and the cDNA employed in the biosensor assembly were optimized by cyclic voltammetry using Fc (CH2OH)2 as a redox probe (Fig. 3). Optimization experiments were performed by incubating the activated GO-CO2H and aptamer/GO-CO2H modified electrodes in different concentrations of the aptamer and the cDNA, respectively. We observed that the redox probe current decreases with the increase of concentrations of each oligonucleotide to level off at 50 μM for both as it can be seen in Fig. 3. Thus, the high amount of the aptamer immobilized on the electrode surface is essential to obtain sensitive detection of the target analyte. For further studies, a concentration of 50 μM of the aptamer and cDNA were used.
The stepwise assembly of the aptasensor was characterized by cyclic voltammetry in presence of Fc(CH2OH)2 (Fig. 4). As described above, we noticed that the coating of electrode with GO-CO2H resulted in an increase of the current intensity, even more remarkable in the cathodic peak. Subsequently, the immobilization of the aptamer provoked the blocking of the electrode surface yielding a substantial reduction of the signal. Furthermore, we remarked that the sequential addition of ethanolamine and cDNA induced small changes in the current suggesting the successful blocking of the surface. Similar results were achieved by electrochemical impedance spectroscopy (EIS). The experimental data were fitted by using an equivalent circuit model (inset of Fig. 4B). EIS plot for the SPCE shows an enlarged semicircle with a diameter of RCT equal to 39.86 kΩ. However, after been coated with GO-CO2H layer, the surface modification induces Fig. 3. Cyclic voltammograms recorded with the GO-CO2H/SPCE in 0.1 M KCl solution containing 1 mM Fc(CH2OH)2 (A) before (a) and after immobilization of the aptamer at different concentrations: 0.5 (b), 5 (c), 10 (d), 50 (e) and 100 μM (f). (B) After the hybridization of the cDNA on anti-PSA aptamer/ GO-CO2H/SPCE using different cDNA concentrations: 0 (a), 1 (b), 10 (c), 50 (d) and 100 μM (e).
3
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Fig. 4. (A) CVs and (B) EIS of SPCE recorded in 0.1 M KCl solution containing 1 mM of Fc(CH2OH)2 before (a) and after sequential modifications with GO-CO2H (b), aptamer immobilization (c), ethanolamine addition (d) and hybridization with the complementary DNA (e).
a dramatic decrease of RCT as revealed in the decrease of the semicircle diameter at high frequencies in the corresponding Nyquist plots (0.96 kΩ). The GO-CO2H film greatly improves the conductivity and the electron transfer process. The RCT slowly increases after the stepwise modification by the aptamer (2.16 kΩ) then the cDNA (10.62 kΩ). The elaboration of the biosensor was also visualized by scanning electron microscopy showing changes in the surface for the different steps (Figure 1S).
single-stranded DNA via favorable π―π stacking interactions with its adjacent base pairs [35]. The MB bound to dsDNA makes it easier to be reduced than when it is linked to ssDNA. The use of such hybridization indicator is a great method to discriminate between ssDNA and dsDNA, which result in potential shift and current variation. This biosensor has the advantage to be amperometric and potentiometric at the same time. A relationship linking the differential pulse voltammogram current to the logarithm of the analyte concentration over five decades of concentrations ranging from 1 pg·mL−1 to 100 ng·mL-1 was established. The linear regression equation was:
3.3. Sensing of the analyte
ΔI (μA) = 0.269 log[PSA] (pg·mL–1) + 3.444
First, we examined the use of ferrocenedimethanol, under the optimized conditions, as a redox probe potentially able to evaluate the PSA biorecognition. However, as it is shown in Fig. 2S, at different PSA concentrations the signal remained unchanged, and hence it is not useful as biosensing approach. As second tentative, we tested the biosensor with ferri/ferrocyanide but we encountered the same behavior of ferrocenedimethanol (Fig. 3S), then we opted for a different approach. The intercalation of MB molecules is well recognized in aptasensing since this organic dye is endowed with redox properties and high affinity to dsDNA [28,30–33]. This dye is reducible to its leucomethylene blue after the exchange of two electrons and one proton reaction at physiological pH [34]. As it can be seen in Fig. 5A, the MB current decreased with the increase of PSA concentration in the range from 1 pg·mL−1 to 500 ng mL−1, suggesting the release of MB and the loosely bound DNA oligonucleotide after the addition of analyte. The MB redox signals shifted to more cathodic potentials, which reflect different environment of MB before and after binding of the target analyte to the active surface. To explain this behavior, we suggest that MB is probably linked to the backbone negatively charged phosphate via electrostatic interactions in the case of single-stranded DNA. Although, MB is more prone to bind to double-stranded DNA than to
The correlation coefficient (R2) is 0.999 and the limit of detection is 0.064 pg·mL−1. This parameter was calculated according to the 3Sb/m criterion, where m is the slope of the calibration curve and Sb was estimated as the standard deviation of three different DPV signals recorded for the lowest analyte concentration measured. A logarithmic relationship was also elucidated between the potential values and the analyte concentration in the same linear range according the following equation: ΔE (V) = 0.01 log[PSA] (pg·mL–1) + 0.163 With a limit of detection equal to 1 pg·mL−1 and a correlation coefficient (R²) of 0.991. The comparison of our biosensor performances with previously reported electrochemical biosensors shows the excellent behavior of our system for PSA detection (Table 2). It allows the detection of the antigen over a wide range of concentrations with the lowest limit of detection.
Fig. 5. (A) Differential pulse voltammograms of MB immobilized in the aptasensing system recorded in 0.1 M PB solution (pH 7.4) after sequential additions of different concentrations of PSA ranging from 1 pg·mL−1 to 500 ng mL−1. (B) Calibration plots of the developed aptasensor for PSA detection. 4
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Table 2 Comparison of some reported biosensors with the present work. Detection system
Method
Range (ng·mL−1)
LOD (ng·mL−1)
reference
Au/apta-MIP GCE/poly(JUG-co-JUGA)/Apta electrode Au/MSF/apta GCE/rGO-MWCNT/AuNPs/apta
capacitance SWV DPV DPV EIS DPV DPV
0.1 to 100 1 to 104 1 to 300 0.005 to 20 0.005 to 100 0.25 to 200 0.001- 100
0.001 1 0.28 0.001
[36] [37] [38] [39]
0.25 0.064 × 10−3
[40] Present work
PGE/AuNP@GMC/apta SPCE/GO-CO2H/apta/cDNA/MB
LOD: limit of detection, Au: gold electrode, Apta: anti-PSA aptamer, MIP: molecularly imprinted polymer, GCE: glassy carbon electrode, p(JUG-co-JUGA): poly(5hydroxy-1,4- naphthoquinone-co- 3-(5-hydroxy-1,4-dihydro-1,4-dioxonaphthalen-2(3)yl)propionic acid), SWV: square wave voltammetry, MSF: Mesoporous silica thin films, rGO: reduced graphene oxide, MWCNT: multi-walled carbon nanotubes, AuNPs: gold nanoparticles, PGE: pyrolytic graphite electrode, AuNP@GMC: gold nanoparticles encapsulated in graphitized mesoporous carbons.
3.5. Stability, reproducibility and applicability in complex matrix The aptasensor stability was studied by following the change in the DPV signal of MB for two weeks for a set of electrodes stored at 4 °C. We observed that biosensors retained ca. 98% of their initial currents showing good stability and detection reliability (Fig. 4S). The reproducibility of the biosensor was studied using three batches of electrodes, each batch contain three electrodes, over three periods of time. We tested them with a 100 pg·mL−1 concentration of PSA. The first batch was taken from the calibration curve of the detection, the second after 8 months and the third after 10 months. The results prove that the system is reproducible in time (Fig. 5S). Since our aptasensor exhibited good selectivity for PSA in presence of the major proteins existing in human blood sera, we ought to confirm this selectivity in more complex samples such as blood sera. We used the device to determine PSA in spiked human sera. The detection process was the same as that in spiked buffered solutions. The blood sera were diluted twice in order to decrease their viscosity. The analytical results were gathered in Table 3. The values of the measured concentrations after successive additions of the analyte to the blood serum solution are close to those added ones. Furthermore, the recovery values are between 99% and 104%, which demonstrate that the method can be reasonably applied for physiological liquids.
Fig. 6. Selectivity (Dark cyan) and specificity (Violet) tests in the presence of 200 pg·mL−1 of each interferent (IgG, CEA and HSA), 130 mM of Na+, 4 mM of K+ and 100 pg·mL−1 of PSA. The error bars are calculated from three independent measurements. Table 3 Determination of PSA in human blood sera using the developed aptasensor. Samples
Added (P) (pg·mL−1)
Found (F) (pg·mL−1)
Recoverya (%)
Human blood sera
0 10 100
ND1 9.92 103.8
– 99 ± 6 104 ± 6
a
4. Conclusions We have successfully developed a novel disposable electrochemical biosensor based on GO-CO2H modified SPCE for rapid and sensitive PSA detection in buffered solutions and complex matrices such as human blood sera. The detection strategy is based on a competitive process between PSA recognized by the aptamer and the DNA strand which hybridized to the aptamer. MB, employed as redox-active indicator, is intercalated between the two DNA strands. Recognition of the antigen results in the release of MB molecules and cDNA, thus the decrease of the DPV signal and the shift of potential to cathodic values. The prepared aptasensor performance shows a sensitive detection of PSA with a good detection limit in 5-decades range of concentrations and it is able to quantify the antigen in complex matrix.
Average of three determinations ± standard deviation, ND: not detected.
3.4. Selectivity and specificity To validate the biosensor analytical performances, we challenged it against three different interferents present in human blood sera. It was found that an excellent discrimination was achieved in presence of HSA, CEA and IgG. PSA gave a neat decrease in current however the other three interferents with 2-fold concentration (200 pg·mL−1) do not provoke any significant change in the currents denoting a very good selectivity toward PSA. Na+ and K+ were also tested with their physiological concentrations. They had a little effect on the current intensity compared to the reaction with PSA (Fig. 6). Specificity of the aptasensor was also investigated. The biosensor was tested by measuring and comparing the response of the as-mentioned interferents together in presence and in absence of the target analyte. No noticeable current change was observed from the mixture without PSA compared to the mixture with PSA. The results indicate a good specificity of the developed biosensing system.
Acknowledgments The authors acknowledge the financial support from the Tunisian Ministry of Higher Education and Scientific Research (lab support for LR99ES15), Spanish Ministry of Economy and Competitiveness (MINECO Projects CTQ2014-58989-P and CTQ2015-71936-REDT). AR thanks the University of Tunis El Manar for the “Bourse d’Alternance” mobility grant and ANEC (ISP-funded network) for the financial support. 5
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Appendix A. Supplementary data [23]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.126762.
[24]
References
[25]
[1] E.T.S.G. da Silva, D.E.P. Souto, J.T.C. Barragan, J.F. Giarola, A.C.M. de Moraes, L.T. Kubota, Electrochemical biosensors in point‐of‐care devices: recent advances and future trends, ChemElectroChem 4 (2017) 778–794. [2] The Refreshed World Cancer Declaration, (2019) (visited 10/11/2018), https:// www.uicc.org/refreshed-world-cancer-declaration. [3] G. Kaur, I. Roy, Therapeutic applications of aptamers, Expert Opin. Investig. Drugs 17 (2008) 43–60. [4] Y. Zhang, Y. Chen, D. Han, I. Ocsoy, W. Tan, Aptamers selected by cell-SELEX for application in cancer studies, Bioanalysis 2 (2010) 907–918. [5] V. Thiviyanathan, D.G. Gorenstein, Aptamers and the next generation of diagnostic reagent, Proteomics Clin. Appl. 6 (2012) 563–573. [6] J.C. Lai, H.E. Horng, C.Y. Hong, Multiplex immunoassays utilizing diferential afnity using aptamers generated by MARAS, Sci. Rep. 7 (2017) 6397. [7] P.B. Crulhas, A.E. Karpik, F.K. Delella, G.R. Castro, V.A. Pedrosa, Electrochemical aptamer-based biosensor developed to monitor PSA and VEGF released by prostate cancer cells, Anal. Bioanal. Chem. 409 (2017) 6771–6780. [8] N. Mottet, J. Bellmunt, M. Bolla, E. Briers, M.G. Cumberbatch, M. De Santis, N. Fossati, T. Gross, A.M. Henry, S. Joniau, T.B. Lam, M.D. Mason, V.B. Matveev, P.C. Moldovan, R.C.N. Van den Bergh, T. Van den Broeck, H.G. Van der Poel, T.H. Van der Kwast, O. Rouviere, I.G. Schots, T. Wiegel, P. Cornford, EAU-ESTROSIOG Guidelines on prostate cancer. Part 1: screening, diagnosis, and local treatment with curative intent, Eur. Urol. 71 (2017) 618–629. [9] E. Heydari-Bafrooeia, N.S. Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosens. Bioelectron. 91 (2017) 284–292. [10] J. Ferlay, E. Steliarova-Foucher, J. Lortet-Tieulent, S. Rosso, J.W.W. Coebergh, H. Comber, D. Forman, F. Bray, Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012, Eur. J. Cancer 49 (2013) 1374–1403. [11] F. Bray, L.A. Kiemeney, Epidemiology of prostate cancer in Europe: patterns, trends and determinants, Management of Prostate Cancer, Springer, 2017, pp. 1–27. [12] Y.-L. Deng, D.-D. Xu, D.-W. Pang, H.-W. Tang, Target-triggered signal turn-on detection of prostate specific antigen based on metal enhanced fluorescence of Ag@ SiO2@SiO2- RuBpy composite nanoparticles, Nanotechnology 28 (2016) 501–510. [13] T.T.N. Do, T.V. Phi, T.P. Nguy, P. Wagner, K. Eersels, M.C. Vestergaard, L.T.N. Truong, Anisotropic in situ-coated aunps on screen-printed carbon surface for enhanced prostate-specific antigen impedimetric aptasensor, J. Electron. Mater. 46 (2017) 3542–3552. [14] R.-M. Kong, L. Ding, Z. Wang, J. You, F. Qu, A novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor for sensitive detection of prostate specific antigen, Anal. Bioanal. Chem. 407 (2015) 369–377. [15] B.-Y. Fang, C.-Y. Wang, C. Li, H.-B. Wang, Y.-D. Zhao, Amplified using DNase I and aptamer/graphene oxide for sensing prostate specific antigen in human serum, Sens. Actuators B Chem. 244 (2017) 928–933. [16] Q. Zhou, Y. Lin, J. Shu, K. Zhang, Z. Yu, D. Tang, Reduced graphene oxide-functionalized FeOOH for signal-on photoelectrochemical sensing of prostate-specific antigen with bioresponsive controlled release system, Biosens. Bioelectron. 98 (2017) 15–21. [17] T. Hao, X. Wu, L. Xu, L. Liu, W. Ma, H. Kuan, C. Xu, Ultrasensitive detection of Prostate-Specific Antigen and Thrombin based on gold-upconversion nanoparticle assembled pyramids, Small 13 (2017) 1603944. [18] M. Bielefeld-Sévigny, AlphaLISA Immunoassays: the no-wash alternative to ELISAs for research and drug discovery, Assay Drug Dev. Technol. 7 (2009) 90–92. [19] B. Kavosi, A. Salimi, R. Hallaj, F. Moradi, Ultrasensitive electrochemical immunosensor for PSA biomarker detection in prostate cancer cells using gold nanoparticles/PAMAM dendrimer loaded with enzyme linked aptamer as integrated triple signal amplification strategy, Biosens. Bioelectron. 74 (2015) 915–923. [20] P. Jolly, V. Tamboli, R.L. Harniman, P. Estrela, C.J. Allender, J.L. Bowen, AptamerMIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen, Biosens. Bioelectron. 75 (2016) 188–195. [21] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: a review, Electroanalysis 22 (2010) 1027. [22] J. Ping, Y. Zhou, Y. Wu, V. Papper, S. Boujday, R.S. Marks, T.W.J. Steele, Recent
[26] [27] [28] [29]
[30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
advances in aptasensors based on graphene and graphene-like nanomaterials, Biosens. Bioelectron. 64 (2015) 373–385. M.S. Artiles, C.S. Rout, T.S. Fisher, Graphene-based hybrid materials and devices for biosensing, Adv. Drug Deliv. Rev. 63 (2011) 1352–1360. A. Tabasi, A. Noorbakhsh, E. Sharifi, Reduced graphene oxide-chitosan-aptamer interface as new platform for ultrasensitive detection of human epidermal growth factor receptor 2, Biosens. Bioelectron. 95 (2017) 117–123. Q. Wang, Z. Zhou, Y. Zhai, L. Zhang, W. Hong, Z. Zhang, S. Dong, Label-free aptamer biosensor for thrombin detection based on functionalized graphene nanocomposites, Talanta 141 (2015) 247–252. S. Deng, V. Tjoa, H.M. Fan, H.R. Tan, D.C. Sayle, M. Olivo, S. Mhaisalkar, J. Wei, C.H. Sow, Reduced graphene oxide conjugated Cu2O nanowire mesocrystals for high-performance NO2 gas sensor, J. Am. Chem. Soc. 134 (2012) 4905–4917. D.-H. Jung, B.H. Kim, Y.K. Ko, M.S. Jung, S. Jung, S.Y. Lee, H.-T. Jung, Covalent attachment and hybridization of DNA oligonucleotides on patterned single-walled carbon nanotube films, Langmuir 20 (2004) 8886–8891. K. Kerman, D. Ozkan, P. Kara, B. Meric, J.J. Gooding, M. Ozsoz, Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes, Anal. Chim. Acta 462 (2002) 39–47. K.Y. Goud, A. Hayat, G. Catanante, M. Satyanarayana, K.V. Gobi, J.L. Marty, An electrochemical aptasensor based on functionalized graphene oxide assisted electrocatalytic signal amplification of methylene blue for aflatoxin B1 detection, Electrochim. Acta 244 (2017) 96–103. S. Nafisi, A.A. Saboury, N. Keramat, J.-F. Neault, H.-A. Tajmir-Riahi, Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue, J. Mol. Struct. 827 (2007) 35–43. A. Erdem, K. Kerman, B. Meric, U.S. Akarca, M. Ozsoz, Novel hybridization indicator methylene blue for the electrochemical detection of short DNA sequences related to the hepatitis B virus, Anal. Chim. Acta 422 (2000) 139–149. T. Hianik, J. Wang, Electrochemical aptasensors – recent achievements and perspectives, Electroanalysis 21 (2009) 1223–1235. E. Farjami, L. Clima, K.V. Gothelfa, E.E. Ferapontova, DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific, Analyst 135 (2010) 1443–1448. H. Ju, J. Zhou, C. Cai, H. Chen, The electrochemical behavior of methylene blue at a microcylinder carbon fiber electrode, Electroanalysis 7 (1995) 1165–1170. H.-A. Rafiee-Pour, M. Behpour, M. Keshavarz, A novel label-free electrochemical miRNA biosensor using methylene blue as redox indicator: application to breast cancer biomarker miRNA-2, Biosens. Bioelecron. 77 (2016) 202–207. P. Jolly, V. Tamboli, R.L. Harniman, P. Estrela, C.J. Allender, J.L. Bowen, Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen, Biosens. Bioelecron. 75 (2016) 188–195. M. Souada, B. Piro, S. Reisberg, G. Anquetin, V. Noël, M.C. Pham, Label-free electrochemical detection of prostate specific antigen based on nucleic acid aptamer, Biosens. Bioelecron. 68 (2015) 49–54. W. Argoubi, A. Sanchez, C. Parrado, N. Raouafi, R. Villalonga, Label-free electrochemical aptasensing platform based on mesoporous silica thin film for the detection of prostate specific antigen, Sens. Actuators B Chem. 255 (2018) 309–315. E. Heydari-Bafrooe, N.S. Shamszadeh, Electrochemical bioassay development for ultrasensitive aptasensing of prostate specific antigen, Biosens. Bioelectron. 91 (2017) 284–292. B. Liu, L. Lu, E. Hua, S. Jiang, G. Xie, Detection of the human prostate-specific antigen using an aptasensor with gold nanoparticles encapsulated by graphitized mesoporous carbon, Microchim. Acta 178 (2012) 163–170.
Amal Raouafi is a Ph.D. student in Chemistry working at the University of Tunis El Manar, Tunisia. Her research is focused on the design of electrochemical biosensors based on carbon nanomaterials for biological applications. Alfredo Sanchez is a Ph.D. in Chemistry. He is currently a Postdoc Researcher at Complutense university of Madrid, Spain. Noureddine Raouafi is currently of professor of Chemistry at the University of Tunis El Manar and director of the Analytical Chemistry and Electrochemistry Laboratory in the as mentioned university. His research interests are devoted to the design of electrochemical nano(bio)sensors for health care, environmental monitoring and food safety. Reynaldo Villalonga is a Professor of Analytical Chemsitry and head of the Nanosensors and Nanomachines group at Complutense University of Madrid, Spain.
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