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polymer composite and gold nanoparticles
Talanta 210 (2020) 120666
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An electrochemical sandwich-type aptasensor for determination of lipocalin2 based on graphene oxide/polymer composite and gold nanoparticles
T
Gözde Aydoğdu Tığ∗, Şule Pekyardımcı Ankara University, Faculty of Science, Department of Chemistry, Ankara, 06100, Turkey
ARTICLE INFO
ABSTRACT
Keywords: Aptasensor Electrochemical Lipocalin-2 Sandwich assay
In this work, we reported an electrochemical aptasensor based on the poly-3-amino-1,2,4-triazole-5-thiol/graphene oxide composite (P(ATT)-GO) and gold nanoparticles (AuNPs) modified graphite screen-printed electrode (GSPE) (GSPE/P(ATT)-GO/AuNPs) for determination of lipocalin-2 (LCN2) (neutrophil gelatinase-associated lipocalin). A sandwich based strategy was utilized to enhance the electrochemical signal. First, a thiol tethered DNA aptamer was immobilized onto the composite electrode. Then, the LCN2 solution was incubated with the aptamer modified GSPE/P(ATT)-GO/AuNPs. Secondary aptamer (Apt2) peculiar to the LCN2 and labeled with biotin was interacted with the LCN2. A streptavidin-alkaline phosphatase conjugate was then applied to the surface. The determination of LCN2 was performed by using the electroactive property of α-naphthol which is acquired the product from the interaction between alkaline phosphatase and α-naphthyl phosphate. The constructed electrode was characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The aptamer modified GSPE/P(ATT)-GO/AuNPs showed the superior electrocatalytic performance towards the voltammetric determination of LCN2 with a wide linear range (1.0–1000.0 ng/mL) and a low limit of detection (LOD) (0.3 ng/ mL). The proposed aptasensor revealed the excellent sensitivity, anti-interference ability and reproducibility which approved that the GSPE/P (ATT)-GO/AuNPs is a promising composite for the sensitive detection of LCN2. The fabricated aptasensor was applied for the determination of LCN2 in fetal bovine serum samples using the standard addition method and the recovery values were in the range of 99.2% and 103.22%.
1. Introduction Lipocalin-2 (LCN2) (neutrophil gelatinase-associated lipocalin) is an essential biomarker for the diagnosis of acute renal failure. It is identified as a 24 kDa secretory glycoprotein in urine and found in neutrophils and renal tubules [1]. The primary function of LCN2 is considered to be related to the transport of small molecules, which are responsible for inflammation, iron metabolism, and apoptosis [2]. LCN2 is found at low concentrations in several human tissues such as colon, lung, stomach, and kidney [3]. The threshold levels for LCN2 in healthy adults is 0.7 ng/mL; however, LCN2 expression can be increased in many disorders such as coronary heart disease [4], bacterial infections [5], kidney transplantation [6], various types of cancer such as ovarian [7], pancreatic [8], and breast [9]. Recently, this protein has been used as an alternative and quantitative biomarker for early diagnosis of hepatocellular carcinoma (HCC) [10,11]. Although the recent developments in therapeutic methods and clinical diagnostics in HCC, the mortality rate for HCC is still very high as most of the patients can
∗
be diagnosed at the last stages [12]. Thus, it is crucial to establish an appropriate diagnostic method for early diagnosis of HCC using a promising biomarker, LCN2. Most previous analytical studies have focused on measuring LCN2 concentration via the enzyme-linked immunosorbent assay (ELISA) [13] and immunoblotting [14]; however, these methods are not cost-effective and require many preparation steps and expensive reagents. Alternatively, electrochemical techniques with their ease of use, fast response and low cost [15] could be regarded as an alternative tool to determine the LCN2 levels in biological samples. Aptamers are short, artificial, and single-stranded DNA or RNA oligonucleotides with high selectivity for various target molecules such as proteins [16], cells [17], small molecules [18,19] and microorganisms [20]. They have been widely used due to their stability, reusability, and ease of modification with functional groups [21]. Therefore, aptamer-based biosensors have been extensively studied in genetic screening, pesticide analysis and diagnostic applications in recent years [22–24]. It is also possible to design novel aptasensors to detect different molecules by using various electrochemical techniques such as
Corresponding author. E-mail address: [email protected] (G. Aydoğdu Tığ).
https://doi.org/10.1016/j.talanta.2019.120666 Received 4 October 2019; Received in revised form 16 December 2019; Accepted 20 December 2019 Available online 23 December 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 210 (2020) 120666
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voltammetric, amperometric, impedimetric and potentiometric [25]. Recently, various nanomaterials such as metal/bimetallic nanoparticles [26], conducting polymers [27], carbon-based nanostructures [28] and their composites [29] have been employed to modify the electrode surface to enhance the electrochemical performance. Mainly, graphene oxide (GO) is considered as a promising material for sensor/ biosensor fabrication due to its unique chemical and physical properties such as large surface area, functional groups, and high stability [30,31]. In the last years, conducting polymer-graphene oxide composites have been used to prevent the agglomeration of GO and to provide a one-step deposition and precise control of the thickness on the electrode surface [32,33]. Thus, the GO-polymer nanocomposite could be useful for novel electrochemical sensing platforms with increasing electrochemical sensitivity. Gold nanoparticles (AuNPs) are extensively preferred to construct various biosensors because of its excellent conductivity, large surface area, easy electrodeposition and biocompatibility [34,35]. In the literature, few studies were reported for the detection of LCN2. An electrochemical immunoassay for the determination of LCN2 using PAMAM dendrimer and AuNPs modified gold electrode was investigated by Kannan et al. [36]. In another study, an immunosensor based on a graphene/poly-aniline modified screen-printed carbon electrode was prepared for the analysis of LCN2 [3]. Thus, the development of sensitive and accurate techniques for the determination of LCN2 in the biological fluid is hugely desirable. To best our knowledge, this is the first study to use the aptamer as biorecognition molecules for the detection of LCN2 biomarker. The novelty of this sensing strategy compared to those reported literature was the use of dual sensing aptamer technology, which was selected according to the literature [11] based on a sandwich assay. In this study, a novel electrochemical aptasensor based on a poly-3amino-1,2,4-triazole-5-thiol/graphene oxide (P(ATT)-GO) and gold nanoparticles (AuNPs) nanocomposite was prepared by two-step electrodeposition method. In order to obtain a sensitive and high specific detection of LCN2, an efficient aptasensor based on a sandwich assay was developed. In this aptasensor, the P(ATT)-GO and AuNPs composite can effectively increase the functional groups, improving electron transfer kinetics and stability. AuNPs could be used to immobilize the thiolated aptamer chain by Au–S bonding. Additionally, the developed aptasensor presented many advantages such as sensitivity, excellent selectivity and good reproducibility to the LCN2 detection. Finally, the proposed aptasensor was applied to fetal bovine serum samples to ascertain the practical utility of the sensing strategy and high recovery values were obtained and compared with the results obtained from a conventional ELISA method.
Operon (Ebersberg, Germany) and listed below: Thiolated Apt1: 5’–(SH)–(CH2)6-CGGAGGGCGGAAGCAAAGCGTAACAGAAAGCCAA CACGCG. Biotinylated Apt2: 3’-(biot)-(CH2)6 -CCACAGTAGGTGAGGTTCACTGAGTTATCCATTG TTGGCA. All oligonucleotide stock solutions were prepared in 10 mmol L−1 TRIS buffer (pH 8.0) and stored at −20 °C.
2. Experimental
The GO-polymer composite was prepared in accordance with the reported procedure in our previous works [26,38]. Briefly, 1.0 mg of GO was dispersed into 1 mL of 1.0 mmol L−1 ATT (0.1 mol L−1 H2SO4) under ultrasonication for 30 min at room temperature, and a homogeneous black suspension was denoted as ATT-GO mixture. To obtain P (ATT)-GO modified GSPE (GSPE/P(ATT)-GO), electropolymerization was achieved using continuous cyclic voltammetry at a scan rate of 50 mV s−1 for 15 cycles in the potential range from −0.20 V and +1.70 V (vs. Ag/AgCl) in ATT-GO mixture under N2 atmosphere [39]. The gold nanoparticles (AuNPs) were electrodeposited on the surface of GSPE/P(ATT)-GO according to a previous report [40]. Briefly, The AuNPs were electrodeposited on the surface of the GSPE/P(ATT)-GO using cyclic voltammetry in 0.5 mol L−1 H2SO4 solution containing 0.6 mmol L−1 HAuCl4 for 15 cycles in the potential range from −0.2 to +1.2 V at a scan rate of 100 mV s−1. Subsequently, the composite electrode was washed with ultrapure water and allowed to dry in the air.
2.2. Instruments Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed on a potentiostat/galvanostat Autolab PGSTAT 302 N electrochemical analyzer using Nova 2.1.2 software (Eco Chemie, The Netherlands). The experimental conditions maintained for differential pulse voltammetric method were: pulse amplitude 50 mV; step potential 3 mV; modulation time 0.02 s; interval time 0.2 s and scan rate 15 mV s−1. Electrochemical impedance spectroscopy (EIS) measurements were performed on a potentiostat/galvanostat Autolab 100 with FRA software. Experiments were carried out by applying a sinusoidal signal of amplitude 10 mV and the frequency in the range of 10 kHz to 0.01 Hz. The fitting of the experimental results accomplished the analysis of the impedance plots to an equivalent circuit by using Nova 1.11 software. The commercial graphite screenprinted electrode (GSPE) with 4 mm of diameter (ref. DRP-C110, DS SPE), supplied by DropSens (Oviedo, Spain), was used as the working electrode. The GSPE includes three parts; a silver pseudo-reference electrode, a graphite counter electrode and a graphite working electrode. A flexible cable (ref. CAC, DropSens) was used to connect the GSPE to the Autolab System. All measurements were carried out at room temperature (23 ± 2 °C). A 60 μL droplet covered the working area was placed for measurements onto GSPEs. The surface morphology of the modified electrodes was carried out with a FEI Nova NanoSEM 650 scanning electron microscope (FEI, The Netherlands). X-ray photoelectron spectroscopy (XPS) measurements were carried out using PHI 5000 Versa Probe spectrometer (Physical Electronics, USA) with a monochromatic Al Kα X-ray source (hυ = 1486.6 eV). The C1s, N1s, S2p, and Au4f XPS spectra were measured with 0.1 eV steps, and multiplex scan fitting was accomplished with Gauss–Lorentzian functions using Origin 9 program. 2.3. Preparation of modified SPEs
2.1. Reagents 3-amino-1,2,4-triazole-5-thiol, sulphuric acid, 6-mercapto hexanol, potassium chloride, magnesium chloride, potassium ferricyanide, potassium ferrocyanide, streptavidin-alkaline phosphatase, diethanolamine, and 1-naphthyl phosphate were purchased from Sigma-Aldrich (Sigma–Aldrich Co.LLC., Germany). Tetrachloroauric acid (HAuCl4·3H2O) was from Acros Organics (Thermo Fischer Scientific, USA). Fetal bovine serum was supplied from Biological Industries Co. (Haemek, Israel). Bovine serum albumin (BSA), human serum albumin (HSA), haptoglobin (HPG), and α-fetoprotein (AFP) (Prestige Antigens) were purchased from Sigma–Aldrich (St. Louis, USA). Graphene oxide (GO) was kindly supplied by Dr. Bülent Zeybek (Dumlupınar University, Department of Chemistry, Turkey). GO was synthesized from graphite powder by a modified Hummers' method according to Ref. [37]. All chemicals and reagents were supplied at analytical grade and used as received without further purification. The DNA aptamer sequences, designed by SELEX, were selected according to a previous report [11] and purchased from Eurofins MWG
2.4. Fabrication of aptasensor The preparation process for aptasensor was illustrated in Scheme 1. 2
Talanta 210 (2020) 120666
G. Aydoğdu Tığ and Ş. Pekyardımcı
Scheme 1. Fabrication steps of the aptasensor for LCN2 detection.
Firstly, the electrodeposition of P (ATT)-GO and AuNPs on the GSPE was performed. Then the modified GSPEs were incubated with 10 μL of 2.0 μmol L−1 thiol-tethered DNA aptamer (Apt 1) for approximately 16 h at +4 °C. The Apt1 modified GSPE/P(ATT)-GO electrodes (GSPE/ P(ATT)-GO/Apt1) were kept in Petri dishes to avoid the evaporation of the solution. Then the GSPE/P(ATT)-GO/Apt1 was washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4 (0.1 mol L−1 NaCl, 0.1 mol L−1 KCl and 5.0 mmol L−1 MgCl2) and 1.0 mmol L−1 of 6mercapto-1-hexanol (MCH) was applied onto the surface of GSPE/P (ATT)-GO/Apt1 for an hour to protect from non-specific interactions. Finally, the electrodes were washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4 for three times. In the next step, the GSPE/P(ATT)-GO/Apt1 was incubated with 10 μL of different concentrations of LCN2 for 30 min at room temperature. Then the obtained GSPE/P(ATT)-GO/Apt1/LCN2 electrode was washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4. The biotinylated secondary aptamer (Apt2) was further applied onto the surface to the GSPE/P(ATT)-GO/Apt1/LCN2 electrode with 10 μL of 1.5 μmol L−1 Apt2 solutions for 45 min. Finally, the aptasensors were washed three times with 20 mmol L−1 TRIS-HCl buffer pH 7.4. The GSPE/P(ATT)-GO/Apt1/LCN2/Apt2 electrode was further incubated with 10 μL of streptavidin-alkaline phosphatase solution (1 U/mL) and 10 mg/mL BSA in 0.1 mol L−1 diethanolamine (DEA) buffer pH 9.6 for 20 min and then it was washed with 0.1 mol L−1 DEA buffer. In the final step, 60 μL of an enzymatic substrate, 1-naphthyl phosphate, solution (1 mg/mL) prepared in 0.1 mol L−1 DEA buffer was dropped onto the aptasensor and waited for 20 min. After the formation of an electroactive product, 1-naphthol, DPV was carried out in the potential range of −0.2 V and +0.35 V, and the voltammetric signal of 1-naphthol at about +0.2 V was used as an analytical response.
2.5. Real sample analysis The fetal bovine serum (FBS) was selected to evaluate the applicability of the proposed aptasensor. The standard addition method was utilized to determine the LCN2 levels in FBS. The standard solutions of LCN2 were added to 1:10 diluted FBS. After aptasensing assay, DPV analysis was carried out and the content of the spiked serum sample was determined from the corresponding regression equation of the calibration plot. The quantitative measurement of LCN2 by ELISA was carried out using Human Lipocalin-2 ELISA Kit (Cat# ab215541, Abcam, ENG) following the manufacturer's instructions. 3. Results and discussion 3.1. Characterization of modified GSPEs SEM image of the GO nanomaterial was obtained and is shown in Fig. S1(A). As seen in the figure, the GO presents a typical flake structure with slight wrinkles. Moreover, EDX analysis has been used for the formation of the GO (Fig. S1(B)). EDX spectrum of GO shows the presence of signals for carbon and oxygen with a carbon weight percentage of 70.32% and an oxygen weight percentage of 24.31%. SEM images of modified electrodes were exhibited in Fig. 1. A thin layer and a spongelike structure were observed when the GSPE surface was modified with P(ATT)-GO (Fig. 1(a)). After AuNPs electrodeposition on the surface of GSPE/P(ATT)-GO electrode (Fig. 1 (b)), well-dispersed particles are seen and indicates the presence of AuNPs. The SEM image of the GSPE/P(ATT)-GO/AuNPs layer shows the presence of the AuNPs with particle sizes of about 65.4 ± 14.5 nm. When the GSPE/P(ATT)GO/AuNPs electrode was incubated with Apt1 (Fig. 1 (c)), it was seen that the roughness between the AuNPs was removed and this indicates
Fig. 1. SEM images of (a) GSPE/P (ATT)-GO, (b) GSPE/P (ATT)-GO/AuNPs and (c) GSPE/P (ATT)-GO/AuNPs/Apt1. 3
Talanta 210 (2020) 120666
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can enhance the electron transfer reaction due to the increased effective surface area. To be specific, such a result can be explained as the following reasons: (i) The abundant oxygen-containing groups in GO and amine group in P(ATT) provide a selective interface for AuNPs deposition. (ii) The π–π stacking interaction and hydrogen bonds between GO and P(ATT) accelerate the electron transfer in the oxidation process, which is responsible for growing peak current. (iii) AuNPs decorated on P(ATT)-GO further enhance the electronic conductivity of the modified electrode and the universal electrocatalytic activity of AuNPs toward most biomolecules is also favourable to the immobilization of aptamer molecules. After the chemisorption of Apt1 onto the GSPE/P(ATT)-GO/ AuNPs surface, the redox peak currents decreased demonstrating the successful immobilization of thiol modified Apt1 with AuNPs (curve d). Electrochemical impedance spectroscopy (EIS) was applied to confirm the modification process of the developed electrode, and the Nyquist plots are given in Fig. 3(B). The semicircle diameter displays the electron transfer resistance value (Ret) at the electrode surface. The bare GSPE showed a semicircle, with a Ret value of 282 Ω. The Ret of the GSPE/P(ATT)-GO decreased to 216 Ω and a remarkable decrease of the Ret value was observed when the GSPE/P (ATT)-GO electrode was modified with the electrodeposited AuNPs (Ret = 33.8 Ω). The results indicate that the electron transfer rate at the solution/electrode interface is increased. However, the Ret value of the GSPE/P(ATT)-GO/ AuNPs/Apt1 electrode increased to 84.7 Ω after Apt1 was immobilized on the surface which is attributed to the negatively charged phosphate backbone of Apt1 that can hinder the electron transfer [46].
Fig. 2. XPS survey spectrum for the GSPE/P (ATT)-GO/AuNPs electrode.
that the Apt1 was successfully attached on the GSPE/P(ATT)-GO/ AuNPs platform. X-ray photoelectron spectroscopy (XPS) was carried out to investigate the elemental analysis of the GSPE/P(ATT)-GO/AuNPs electrode. As can be seen in Fig. 2, the survey spectra showed that C1s, N1s, O1s, S2p, Au4p3, Au4d3, Au4d5, Au4f5, and Au4f7 peaks were determined on the surface of the GSPE/P(ATT)-GO/AuNPs electrode. In the expanded spectrum of C1s (Fig. S2(A)), the first peak at about 282.91 eV is due to C–C/C]C and C–H bonds, the second one with binding energy of 284.33 eV is attributed to the C–N/C–O bonds, and the third peak at 287.02 eV is related to the C]N/O–C]O bonds [41]. The N1s spectrum (Fig. S2(B)) exhibits two peaks with binding energies at 398.04 eV and 399.89 eV, which can be ascribed to C–N/C]N and N–H bonds [42], respectively. These results indicate the formation of P (ATT)-GO composite on the GSPE surface. The high-resolution S2p spectrum (Fig. S2(C)) displays two peaks centered at 160.3 eV and 167.02 eV. The peak at 160.3 eV can be assigned to the S–S bond and the peak at 167.02 eV can be attributed to the doped sulfate ion [43]. XPS expanded spectrum of Au4f exhibited the Au4f7/2 and Au4f5/2 doublet with binding energies of 82.11 eV and 85.78 eV, respectively. The presence of these peaks indicates that the HAuCl4 was successfully reduced to metallic gold [44].
3.2. Optimum conditions for aptasensor To obtain the best conditions for the developed aptasensor, several experimental factors were optimized. The effect of Apt1 concentration on the response signal was investigated in the range from 0.5 μmol L−1 to 5.0 μmol L−1. Fig. 4(A) shows the voltammetric signal of α-naphthol at the 50.0 ng/mL of the LCN2 concentration with the increasing amount of Apt1 concentration. As can be seen in Fig. 4(A) (inset) the current signal increased up to 1.0 μmol L−1 Apt1 and then leveled off. Thus, the optimum Apt1 concentration was chosen as 1.0 μmol L−1 in the subsequent experiments. The effect of the Apt2 concentration on the response of the modified electrode was studied between 0.5 μmol L−1 to 5.0 μmol L−1. As shown in Fig. 4(B), The maximum current value was obtained at 1.5 μmol L−1 Apt2 concentration. Therefore, the optimum Apt2 concentration was selected as 1.5 μmol L−1. The incubation time is another important parameter that affects the performance of the aptasensor. The experiments to optimize the incubation time were performed incubating the aptasensor with a 50.0 ng/mL LCN2 and 1.5 μmol L−1 Apt2 at different times (Fig. 5). When the incubation time increased, the current signal increased until 30 min and then tended to reach saturation behavior because the aptamers completely interacted with LCN2 molecules. It was suggested that 30 min was the optimum incubation time for the developed aptasensor.
3.1.1. Optimization of number of potential cycles on the response of LCN2 To investigate the effect of P(ATT)-GO thickness on the current response of LCN2, the different number of potential cycles during the electrodeposition were studied. The P(ATT)-GO was electropolymerized on the GSPE in 0.1 mol L−1 H2SO4 containing 1 mg/mL of GO and 1.0 mmol L−1 ATT using cycles ranging from 2 to 30. As can be seen in Fig. S3, the peak current of LCN2 increased with the increasing number of cycles to 15. The current response slightly decreased with the further increase in the number of potential cycles suggesting that the thicker polymer film could generate a barrier for electron transfer reaction [45]. Thus, the electropolymerization cycles of 15 were chosen to construct the aptasensor in the subsequent experiments. The electrochemical characterization of each modification step was monitored by CV and EIS. Fig. 3(A) shows the CVs of the different modified GPSEs in 0.1 mol L−1 KCl containing 5.0 mmol L−1 Fe (CN)63−/4‒. Bare GSPE exhibited a well-defined redox peak couple (curve a) which corresponds to the reversible redox reaction of Fe (CN)63−/4‒. After the electrodeposition of P(ATT)-GO composite, a remarkable increase in redox peak currents was observed due to the conductive properties of P(ATT)-GO layer (curve b). With the modification of AuNPs onto the GSPE/P(ATT)-GO electrode, the highest redox peak currents are observed which can be ascribed to the good conductivity of the AuNPs (curve c). The GSPE/P(ATT)-GO electrode
3.3. LCN2 determination The voltammetric determination of LCN2 was carried out using the different concentrations of the LCN2. As shown in Fig. 6(A), the current signals of an electroactive product, α-naphthol, increased with increasing concentrations of LCN2 Fig. 6(B). The increase in the peak current with each addition of LCN2 is attributed to the formation of an aptamer-LCN2 complex. The fabricated aptasensor exhibited two linear relationships between the current signal and LCN2 concentration; one from 1.0 ng/mL to 50.0 ng/mL with a determination coefficient (R2) of 0.998 (Fig. 6(C)) and another one from 50.0 ng/mL to 1000 ng/mL with a R2 of 0.981 (Fig. 6(D)). The limit of detection (LOD) and the limit of quantification (LOQ) for LCN2 were calculated according to the following equations and found as 0.3 ng/mL and 1.0 ng/mL, respectively. 4
Talanta 210 (2020) 120666
G. Aydoğdu Tığ and Ş. Pekyardımcı
Fig. 3. (A) CV and (B) EIS of (a) bare GSPE, (b) GSPE/P(ATT)-GO, (c) GSPE/P(ATT)-GO/AuNPs, (d) GSPE/P(ATT)-GO/AuNPs/Apt1 electrodes in 0.1 mol L−1 KCl solution containing 5.0 mmol L−1 Fe(CN)63−/4‒.
LOD = 3s / m , LOQ = 10s / m
(1)
Table 1 summarizes the results of previously reported LCN2 sensing platforms in terms of the linear ranges and detection limits with our developed aptasensor. As demonstrated above, the fabricated aptasensor exhibited the improvement in the detection limit compared to the other electrodes given in Table 1 [3,36,47]. This might be attributed to the excellent electrical conductivity, high surface area of the fabricated matrix, and good electron communication obtained by the prepared nanocomposite. 3.4. Selectivity and reproducibility To examine the selectivity of the proposed aptasensor for the LCN2 analysis, bovine serum albumin (BSA), human serum albumin (HSA), haptoglobin (HPG), and α-fetoprotein (AFP) as interfering substances were tested under the same conditions. As shown in Fig. 7, the effect caused by the interfering proteins (at the concentration of 500 ng/mL) using GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2 electrode was negligible with the relative standard deviation (RSD) less than 5.0%. Therefore, it can be concluded that the interaction between aptamer molecules and LCN2 is based on specific recognition. Thus, these observations suggest that the sensing mechanism based on the sandwich method is highly specific [48]. Thus, the prepared aptasensor is highly selective for LCN2 determination. The reproducibility of the aptasensor was evaluated by measuring the analytical response of five different electrodes prepared independently under the same conditions. The RSD% results (1.24%) of the DPV peak current signals prove that the aptasensor has good reproducibility.
Fig. 5. The effect of incubation time on the DPV signal of the α-naphthol at the GSPE/P(ATT)-GO/AuNPs. Inset: Current vs. incubation time of LCN2 (n = 3).
3.5. Real sample analysis To further evaluate the performance of the proposed GSPE/P(ATT)GO/Apt1/LCN2/Apt2 electrode, DPV measurements were performed using a fetal bovine serum (FBS). The recovery tests were carried out by standard additions of LCN2 to FBS samples, and the results are in the range from 99.20% and %103.22, and the RSD values were found to be in the range from 1.17% to 4.35% (Table 2). These results obtained for repeatable on each sample indicate the feasibility of the proposed aptasensor for the determination of LCN2 in fetal bovine serum samples. The accuracy of the developed aptasensor was studied via comparison with a commercially available ELISA kit for the LCN2 determination and checked by a t-test. The RSD values were found to be
Fig. 4. The effect of Apt1(A) and Apt2 (B) concentration on the DPV signal of the α-naphthol at the GSPE/P (ATT)-GO/AuNPs. Inset: Current vs. aptamer concentrations (n = 3). 5
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G. Aydoğdu Tığ and Ş. Pekyardımcı
Fig. 6. (A) Differential pulse voltammograms of various concentrations of LCN2. (B) Effect of LCN2 concentration on the voltammetric response of GSPE/P(ATT)GO/AuNPs/Apt1/LCN2/Apt2 electrode. (C), (D) Linear dependence of LCN2 concentration on the α-naphthol signals at GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2 electrode (n = 3). Table 1 Comparison of analytical performances of the different electrochemical sensors for the determination of LCN2. Electrode
Linearity
LOD
Reproducibility
Application
Reference
Aniline functionalized G/PANI nanodroplet-modified electrode LA2/AuNPs/PAMAM-modified gold electrode
50.0–500.0 ng/mL
21.1 ng/mL 1.0 ng/mL
ITO/TiO2/CoPc/CS/SA/BSA/BiNb/NGAL photoelectrochemical sensor GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2
1.0–500.0 pg/mL
0.6 pg/mL
RSD: 4.7%
1.0–1000.0 ng/mL
0.3 ng/mL
RSD: 1.24% (n = 5)
Human urine Recoveries:97.5–104.4% Human serum Recoveries:98.5–98.8% Human urine Recoveries:96.1–97.6% Human serum Recoveries: 93.2–112% Fetal bovin serum Recoveries: 99.20–103.22
[3]
50.0–250.0 ng/mL
RSD: 1.49–9.20% (n = 5) RSD: 2.1% (n = 10)
[36]
[47] This study
Graphene (G), polyaniline (PANI), rabbit polygonal lipocalin-2 antibody (LA2), gold nanoparticles (AuNPS), generation-1 polyamidoamine (PAMAM), indium tin oxide (ITO), titanium dioxide (TiO2), streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc), chitosan (CS), streptavidin (SA), Bovine serum albumin (BSA), biotinylated nanobodies (BiNb), neutrophil gelatinase-associated lipocalin (NGAL), relative standard deviation (RSD).
less than 5.0% confirming good accuracy of the proposed sensing strategy (Table 3). The t values of three different LCN2 concentrations were found as 0.96, 0.80 and 0.47 at 95% confidence level for tcritic 2.13. The results obtained from our developed aptasensor are in good agreement with the results obtained from the ELISA method, as exhibited in Table 3.
be prepared via a simple electrodeposition procedure. Surface coverage and electrocatalytic properties of the fabricated GSPE/P(ATT)-GO/ AuNPs were remarkably improved in comparison with unmodified electrode. The developed aptasensor showed high sensitivity, selectivity and low LOD (0.3 ng/mL) value using the DPV technique. Moreover, the feasibility of the sensing strategy was investigated in fetal bovine serum samples using the standard addition method and high recovery values (99.20%–103.22%) were obtained with low RSDs. Importantly, the LCN2 levels measured by DPV technique were compared to a conventional ELISA method and the developed aptasensor presented more effortless preparation procedure, high selectivity and accuracy, rapid analysis, and low-cost sensing with a good correlation. Therefore, this sensing platform could be an efficient tool for the determination of LCN2 levels in clinical research.
4. Conclusion Herein, we prepared a novel aptasensor for the determination of LCN2 based on a sandwich assay. The proposed disposable electrode was easy-to-use, cost-effective and required a low amount of reagents. The modified GSPE was obtained by electrodeposition of graphene oxide-poly-3-amino-1,2,4-triazole-5-thiol composite (GO-P(ATT)) and gold nanoparticles (AuNPs). The characterization of the fabricated aptasensor was carried out by using SEM, XPS, CV and EIS techniques. The prepared electrode has suitable reproducibility and stability and could 6
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G. Aydoğdu Tığ and Ş. Pekyardımcı
[4]
[5] [6] [7]
[8]
[9]
Fig. 7. Selectivity of the proposed aptasensor to 50 ng/mL of LCN2 by comparing it to the various proteins at the 500 ng/mL level.
[10]
Table 2 Results of real sample analysis at different concentrations of LCN2. Sample
Added (ng/mL)
Found (ng/mL)
Recovery%
RSD%a
Fetal bovine serum
5.0 10.0 100.0
4.96 10.18 103.22
99.20 101.80 103.22
2.49 1.17 4.35
a
[11] [12] [13]
Each value is the mean of three measurements. [14]
Table 3 Comparison of real sample analysis results with ELISA method. Sample
Developed aptasensor (ng/mL)
ELISA (ng/ mL)
RSD%a (ELISA)
t
Fetal bovin serum
4.96 10.18 100.22
5.12 9.89 99.47
0.89 2.57 3.42
0.96 0.80 0.47
a
[15] [16] [17]
Each value is the mean of three measurements.
Declaration of competing interest
[18]
We certify that there are no conflicts of interest with any financial organization regarding the material discussed in the manuscript. This study was supported by the scientific and technological research council of Turkey (TUBITAK) under grants 116Z846.
[19]
Acknowledgments
[21]
The authors acknowledge the support provided by the scientific and technological research council of Turkey (TUBITAK) under grants 116Z846.
[22]
[20]
[23] [24]
Appendix A. Supplementary data
[25]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120666.
[26]
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An electrochemical sandwich-type aptasensor for determination of lipocalin2 based on graphene oxide/polymer composite and gold nanoparticles
T
Gözde Aydoğdu Tığ∗, Şule Pekyardımcı Ankara University, Faculty of Science, Department of Chemistry, Ankara, 06100, Turkey
ARTICLE INFO
ABSTRACT
Keywords: Aptasensor Electrochemical Lipocalin-2 Sandwich assay
In this work, we reported an electrochemical aptasensor based on the poly-3-amino-1,2,4-triazole-5-thiol/graphene oxide composite (P(ATT)-GO) and gold nanoparticles (AuNPs) modified graphite screen-printed electrode (GSPE) (GSPE/P(ATT)-GO/AuNPs) for determination of lipocalin-2 (LCN2) (neutrophil gelatinase-associated lipocalin). A sandwich based strategy was utilized to enhance the electrochemical signal. First, a thiol tethered DNA aptamer was immobilized onto the composite electrode. Then, the LCN2 solution was incubated with the aptamer modified GSPE/P(ATT)-GO/AuNPs. Secondary aptamer (Apt2) peculiar to the LCN2 and labeled with biotin was interacted with the LCN2. A streptavidin-alkaline phosphatase conjugate was then applied to the surface. The determination of LCN2 was performed by using the electroactive property of α-naphthol which is acquired the product from the interaction between alkaline phosphatase and α-naphthyl phosphate. The constructed electrode was characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The aptamer modified GSPE/P(ATT)-GO/AuNPs showed the superior electrocatalytic performance towards the voltammetric determination of LCN2 with a wide linear range (1.0–1000.0 ng/mL) and a low limit of detection (LOD) (0.3 ng/ mL). The proposed aptasensor revealed the excellent sensitivity, anti-interference ability and reproducibility which approved that the GSPE/P (ATT)-GO/AuNPs is a promising composite for the sensitive detection of LCN2. The fabricated aptasensor was applied for the determination of LCN2 in fetal bovine serum samples using the standard addition method and the recovery values were in the range of 99.2% and 103.22%.
1. Introduction Lipocalin-2 (LCN2) (neutrophil gelatinase-associated lipocalin) is an essential biomarker for the diagnosis of acute renal failure. It is identified as a 24 kDa secretory glycoprotein in urine and found in neutrophils and renal tubules [1]. The primary function of LCN2 is considered to be related to the transport of small molecules, which are responsible for inflammation, iron metabolism, and apoptosis [2]. LCN2 is found at low concentrations in several human tissues such as colon, lung, stomach, and kidney [3]. The threshold levels for LCN2 in healthy adults is 0.7 ng/mL; however, LCN2 expression can be increased in many disorders such as coronary heart disease [4], bacterial infections [5], kidney transplantation [6], various types of cancer such as ovarian [7], pancreatic [8], and breast [9]. Recently, this protein has been used as an alternative and quantitative biomarker for early diagnosis of hepatocellular carcinoma (HCC) [10,11]. Although the recent developments in therapeutic methods and clinical diagnostics in HCC, the mortality rate for HCC is still very high as most of the patients can
∗
be diagnosed at the last stages [12]. Thus, it is crucial to establish an appropriate diagnostic method for early diagnosis of HCC using a promising biomarker, LCN2. Most previous analytical studies have focused on measuring LCN2 concentration via the enzyme-linked immunosorbent assay (ELISA) [13] and immunoblotting [14]; however, these methods are not cost-effective and require many preparation steps and expensive reagents. Alternatively, electrochemical techniques with their ease of use, fast response and low cost [15] could be regarded as an alternative tool to determine the LCN2 levels in biological samples. Aptamers are short, artificial, and single-stranded DNA or RNA oligonucleotides with high selectivity for various target molecules such as proteins [16], cells [17], small molecules [18,19] and microorganisms [20]. They have been widely used due to their stability, reusability, and ease of modification with functional groups [21]. Therefore, aptamer-based biosensors have been extensively studied in genetic screening, pesticide analysis and diagnostic applications in recent years [22–24]. It is also possible to design novel aptasensors to detect different molecules by using various electrochemical techniques such as
Corresponding author. E-mail address: [email protected] (G. Aydoğdu Tığ).
https://doi.org/10.1016/j.talanta.2019.120666 Received 4 October 2019; Received in revised form 16 December 2019; Accepted 20 December 2019 Available online 23 December 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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voltammetric, amperometric, impedimetric and potentiometric [25]. Recently, various nanomaterials such as metal/bimetallic nanoparticles [26], conducting polymers [27], carbon-based nanostructures [28] and their composites [29] have been employed to modify the electrode surface to enhance the electrochemical performance. Mainly, graphene oxide (GO) is considered as a promising material for sensor/ biosensor fabrication due to its unique chemical and physical properties such as large surface area, functional groups, and high stability [30,31]. In the last years, conducting polymer-graphene oxide composites have been used to prevent the agglomeration of GO and to provide a one-step deposition and precise control of the thickness on the electrode surface [32,33]. Thus, the GO-polymer nanocomposite could be useful for novel electrochemical sensing platforms with increasing electrochemical sensitivity. Gold nanoparticles (AuNPs) are extensively preferred to construct various biosensors because of its excellent conductivity, large surface area, easy electrodeposition and biocompatibility [34,35]. In the literature, few studies were reported for the detection of LCN2. An electrochemical immunoassay for the determination of LCN2 using PAMAM dendrimer and AuNPs modified gold electrode was investigated by Kannan et al. [36]. In another study, an immunosensor based on a graphene/poly-aniline modified screen-printed carbon electrode was prepared for the analysis of LCN2 [3]. Thus, the development of sensitive and accurate techniques for the determination of LCN2 in the biological fluid is hugely desirable. To best our knowledge, this is the first study to use the aptamer as biorecognition molecules for the detection of LCN2 biomarker. The novelty of this sensing strategy compared to those reported literature was the use of dual sensing aptamer technology, which was selected according to the literature [11] based on a sandwich assay. In this study, a novel electrochemical aptasensor based on a poly-3amino-1,2,4-triazole-5-thiol/graphene oxide (P(ATT)-GO) and gold nanoparticles (AuNPs) nanocomposite was prepared by two-step electrodeposition method. In order to obtain a sensitive and high specific detection of LCN2, an efficient aptasensor based on a sandwich assay was developed. In this aptasensor, the P(ATT)-GO and AuNPs composite can effectively increase the functional groups, improving electron transfer kinetics and stability. AuNPs could be used to immobilize the thiolated aptamer chain by Au–S bonding. Additionally, the developed aptasensor presented many advantages such as sensitivity, excellent selectivity and good reproducibility to the LCN2 detection. Finally, the proposed aptasensor was applied to fetal bovine serum samples to ascertain the practical utility of the sensing strategy and high recovery values were obtained and compared with the results obtained from a conventional ELISA method.
Operon (Ebersberg, Germany) and listed below: Thiolated Apt1: 5’–(SH)–(CH2)6-CGGAGGGCGGAAGCAAAGCGTAACAGAAAGCCAA CACGCG. Biotinylated Apt2: 3’-(biot)-(CH2)6 -CCACAGTAGGTGAGGTTCACTGAGTTATCCATTG TTGGCA. All oligonucleotide stock solutions were prepared in 10 mmol L−1 TRIS buffer (pH 8.0) and stored at −20 °C.
2. Experimental
The GO-polymer composite was prepared in accordance with the reported procedure in our previous works [26,38]. Briefly, 1.0 mg of GO was dispersed into 1 mL of 1.0 mmol L−1 ATT (0.1 mol L−1 H2SO4) under ultrasonication for 30 min at room temperature, and a homogeneous black suspension was denoted as ATT-GO mixture. To obtain P (ATT)-GO modified GSPE (GSPE/P(ATT)-GO), electropolymerization was achieved using continuous cyclic voltammetry at a scan rate of 50 mV s−1 for 15 cycles in the potential range from −0.20 V and +1.70 V (vs. Ag/AgCl) in ATT-GO mixture under N2 atmosphere [39]. The gold nanoparticles (AuNPs) were electrodeposited on the surface of GSPE/P(ATT)-GO according to a previous report [40]. Briefly, The AuNPs were electrodeposited on the surface of the GSPE/P(ATT)-GO using cyclic voltammetry in 0.5 mol L−1 H2SO4 solution containing 0.6 mmol L−1 HAuCl4 for 15 cycles in the potential range from −0.2 to +1.2 V at a scan rate of 100 mV s−1. Subsequently, the composite electrode was washed with ultrapure water and allowed to dry in the air.
2.2. Instruments Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed on a potentiostat/galvanostat Autolab PGSTAT 302 N electrochemical analyzer using Nova 2.1.2 software (Eco Chemie, The Netherlands). The experimental conditions maintained for differential pulse voltammetric method were: pulse amplitude 50 mV; step potential 3 mV; modulation time 0.02 s; interval time 0.2 s and scan rate 15 mV s−1. Electrochemical impedance spectroscopy (EIS) measurements were performed on a potentiostat/galvanostat Autolab 100 with FRA software. Experiments were carried out by applying a sinusoidal signal of amplitude 10 mV and the frequency in the range of 10 kHz to 0.01 Hz. The fitting of the experimental results accomplished the analysis of the impedance plots to an equivalent circuit by using Nova 1.11 software. The commercial graphite screenprinted electrode (GSPE) with 4 mm of diameter (ref. DRP-C110, DS SPE), supplied by DropSens (Oviedo, Spain), was used as the working electrode. The GSPE includes three parts; a silver pseudo-reference electrode, a graphite counter electrode and a graphite working electrode. A flexible cable (ref. CAC, DropSens) was used to connect the GSPE to the Autolab System. All measurements were carried out at room temperature (23 ± 2 °C). A 60 μL droplet covered the working area was placed for measurements onto GSPEs. The surface morphology of the modified electrodes was carried out with a FEI Nova NanoSEM 650 scanning electron microscope (FEI, The Netherlands). X-ray photoelectron spectroscopy (XPS) measurements were carried out using PHI 5000 Versa Probe spectrometer (Physical Electronics, USA) with a monochromatic Al Kα X-ray source (hυ = 1486.6 eV). The C1s, N1s, S2p, and Au4f XPS spectra were measured with 0.1 eV steps, and multiplex scan fitting was accomplished with Gauss–Lorentzian functions using Origin 9 program. 2.3. Preparation of modified SPEs
2.1. Reagents 3-amino-1,2,4-triazole-5-thiol, sulphuric acid, 6-mercapto hexanol, potassium chloride, magnesium chloride, potassium ferricyanide, potassium ferrocyanide, streptavidin-alkaline phosphatase, diethanolamine, and 1-naphthyl phosphate were purchased from Sigma-Aldrich (Sigma–Aldrich Co.LLC., Germany). Tetrachloroauric acid (HAuCl4·3H2O) was from Acros Organics (Thermo Fischer Scientific, USA). Fetal bovine serum was supplied from Biological Industries Co. (Haemek, Israel). Bovine serum albumin (BSA), human serum albumin (HSA), haptoglobin (HPG), and α-fetoprotein (AFP) (Prestige Antigens) were purchased from Sigma–Aldrich (St. Louis, USA). Graphene oxide (GO) was kindly supplied by Dr. Bülent Zeybek (Dumlupınar University, Department of Chemistry, Turkey). GO was synthesized from graphite powder by a modified Hummers' method according to Ref. [37]. All chemicals and reagents were supplied at analytical grade and used as received without further purification. The DNA aptamer sequences, designed by SELEX, were selected according to a previous report [11] and purchased from Eurofins MWG
2.4. Fabrication of aptasensor The preparation process for aptasensor was illustrated in Scheme 1. 2
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Scheme 1. Fabrication steps of the aptasensor for LCN2 detection.
Firstly, the electrodeposition of P (ATT)-GO and AuNPs on the GSPE was performed. Then the modified GSPEs were incubated with 10 μL of 2.0 μmol L−1 thiol-tethered DNA aptamer (Apt 1) for approximately 16 h at +4 °C. The Apt1 modified GSPE/P(ATT)-GO electrodes (GSPE/ P(ATT)-GO/Apt1) were kept in Petri dishes to avoid the evaporation of the solution. Then the GSPE/P(ATT)-GO/Apt1 was washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4 (0.1 mol L−1 NaCl, 0.1 mol L−1 KCl and 5.0 mmol L−1 MgCl2) and 1.0 mmol L−1 of 6mercapto-1-hexanol (MCH) was applied onto the surface of GSPE/P (ATT)-GO/Apt1 for an hour to protect from non-specific interactions. Finally, the electrodes were washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4 for three times. In the next step, the GSPE/P(ATT)-GO/Apt1 was incubated with 10 μL of different concentrations of LCN2 for 30 min at room temperature. Then the obtained GSPE/P(ATT)-GO/Apt1/LCN2 electrode was washed with 20 mmol L−1 TRIS-HCl buffer pH 7.4. The biotinylated secondary aptamer (Apt2) was further applied onto the surface to the GSPE/P(ATT)-GO/Apt1/LCN2 electrode with 10 μL of 1.5 μmol L−1 Apt2 solutions for 45 min. Finally, the aptasensors were washed three times with 20 mmol L−1 TRIS-HCl buffer pH 7.4. The GSPE/P(ATT)-GO/Apt1/LCN2/Apt2 electrode was further incubated with 10 μL of streptavidin-alkaline phosphatase solution (1 U/mL) and 10 mg/mL BSA in 0.1 mol L−1 diethanolamine (DEA) buffer pH 9.6 for 20 min and then it was washed with 0.1 mol L−1 DEA buffer. In the final step, 60 μL of an enzymatic substrate, 1-naphthyl phosphate, solution (1 mg/mL) prepared in 0.1 mol L−1 DEA buffer was dropped onto the aptasensor and waited for 20 min. After the formation of an electroactive product, 1-naphthol, DPV was carried out in the potential range of −0.2 V and +0.35 V, and the voltammetric signal of 1-naphthol at about +0.2 V was used as an analytical response.
2.5. Real sample analysis The fetal bovine serum (FBS) was selected to evaluate the applicability of the proposed aptasensor. The standard addition method was utilized to determine the LCN2 levels in FBS. The standard solutions of LCN2 were added to 1:10 diluted FBS. After aptasensing assay, DPV analysis was carried out and the content of the spiked serum sample was determined from the corresponding regression equation of the calibration plot. The quantitative measurement of LCN2 by ELISA was carried out using Human Lipocalin-2 ELISA Kit (Cat# ab215541, Abcam, ENG) following the manufacturer's instructions. 3. Results and discussion 3.1. Characterization of modified GSPEs SEM image of the GO nanomaterial was obtained and is shown in Fig. S1(A). As seen in the figure, the GO presents a typical flake structure with slight wrinkles. Moreover, EDX analysis has been used for the formation of the GO (Fig. S1(B)). EDX spectrum of GO shows the presence of signals for carbon and oxygen with a carbon weight percentage of 70.32% and an oxygen weight percentage of 24.31%. SEM images of modified electrodes were exhibited in Fig. 1. A thin layer and a spongelike structure were observed when the GSPE surface was modified with P(ATT)-GO (Fig. 1(a)). After AuNPs electrodeposition on the surface of GSPE/P(ATT)-GO electrode (Fig. 1 (b)), well-dispersed particles are seen and indicates the presence of AuNPs. The SEM image of the GSPE/P(ATT)-GO/AuNPs layer shows the presence of the AuNPs with particle sizes of about 65.4 ± 14.5 nm. When the GSPE/P(ATT)GO/AuNPs electrode was incubated with Apt1 (Fig. 1 (c)), it was seen that the roughness between the AuNPs was removed and this indicates
Fig. 1. SEM images of (a) GSPE/P (ATT)-GO, (b) GSPE/P (ATT)-GO/AuNPs and (c) GSPE/P (ATT)-GO/AuNPs/Apt1. 3
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can enhance the electron transfer reaction due to the increased effective surface area. To be specific, such a result can be explained as the following reasons: (i) The abundant oxygen-containing groups in GO and amine group in P(ATT) provide a selective interface for AuNPs deposition. (ii) The π–π stacking interaction and hydrogen bonds between GO and P(ATT) accelerate the electron transfer in the oxidation process, which is responsible for growing peak current. (iii) AuNPs decorated on P(ATT)-GO further enhance the electronic conductivity of the modified electrode and the universal electrocatalytic activity of AuNPs toward most biomolecules is also favourable to the immobilization of aptamer molecules. After the chemisorption of Apt1 onto the GSPE/P(ATT)-GO/ AuNPs surface, the redox peak currents decreased demonstrating the successful immobilization of thiol modified Apt1 with AuNPs (curve d). Electrochemical impedance spectroscopy (EIS) was applied to confirm the modification process of the developed electrode, and the Nyquist plots are given in Fig. 3(B). The semicircle diameter displays the electron transfer resistance value (Ret) at the electrode surface. The bare GSPE showed a semicircle, with a Ret value of 282 Ω. The Ret of the GSPE/P(ATT)-GO decreased to 216 Ω and a remarkable decrease of the Ret value was observed when the GSPE/P (ATT)-GO electrode was modified with the electrodeposited AuNPs (Ret = 33.8 Ω). The results indicate that the electron transfer rate at the solution/electrode interface is increased. However, the Ret value of the GSPE/P(ATT)-GO/ AuNPs/Apt1 electrode increased to 84.7 Ω after Apt1 was immobilized on the surface which is attributed to the negatively charged phosphate backbone of Apt1 that can hinder the electron transfer [46].
Fig. 2. XPS survey spectrum for the GSPE/P (ATT)-GO/AuNPs electrode.
that the Apt1 was successfully attached on the GSPE/P(ATT)-GO/ AuNPs platform. X-ray photoelectron spectroscopy (XPS) was carried out to investigate the elemental analysis of the GSPE/P(ATT)-GO/AuNPs electrode. As can be seen in Fig. 2, the survey spectra showed that C1s, N1s, O1s, S2p, Au4p3, Au4d3, Au4d5, Au4f5, and Au4f7 peaks were determined on the surface of the GSPE/P(ATT)-GO/AuNPs electrode. In the expanded spectrum of C1s (Fig. S2(A)), the first peak at about 282.91 eV is due to C–C/C]C and C–H bonds, the second one with binding energy of 284.33 eV is attributed to the C–N/C–O bonds, and the third peak at 287.02 eV is related to the C]N/O–C]O bonds [41]. The N1s spectrum (Fig. S2(B)) exhibits two peaks with binding energies at 398.04 eV and 399.89 eV, which can be ascribed to C–N/C]N and N–H bonds [42], respectively. These results indicate the formation of P (ATT)-GO composite on the GSPE surface. The high-resolution S2p spectrum (Fig. S2(C)) displays two peaks centered at 160.3 eV and 167.02 eV. The peak at 160.3 eV can be assigned to the S–S bond and the peak at 167.02 eV can be attributed to the doped sulfate ion [43]. XPS expanded spectrum of Au4f exhibited the Au4f7/2 and Au4f5/2 doublet with binding energies of 82.11 eV and 85.78 eV, respectively. The presence of these peaks indicates that the HAuCl4 was successfully reduced to metallic gold [44].
3.2. Optimum conditions for aptasensor To obtain the best conditions for the developed aptasensor, several experimental factors were optimized. The effect of Apt1 concentration on the response signal was investigated in the range from 0.5 μmol L−1 to 5.0 μmol L−1. Fig. 4(A) shows the voltammetric signal of α-naphthol at the 50.0 ng/mL of the LCN2 concentration with the increasing amount of Apt1 concentration. As can be seen in Fig. 4(A) (inset) the current signal increased up to 1.0 μmol L−1 Apt1 and then leveled off. Thus, the optimum Apt1 concentration was chosen as 1.0 μmol L−1 in the subsequent experiments. The effect of the Apt2 concentration on the response of the modified electrode was studied between 0.5 μmol L−1 to 5.0 μmol L−1. As shown in Fig. 4(B), The maximum current value was obtained at 1.5 μmol L−1 Apt2 concentration. Therefore, the optimum Apt2 concentration was selected as 1.5 μmol L−1. The incubation time is another important parameter that affects the performance of the aptasensor. The experiments to optimize the incubation time were performed incubating the aptasensor with a 50.0 ng/mL LCN2 and 1.5 μmol L−1 Apt2 at different times (Fig. 5). When the incubation time increased, the current signal increased until 30 min and then tended to reach saturation behavior because the aptamers completely interacted with LCN2 molecules. It was suggested that 30 min was the optimum incubation time for the developed aptasensor.
3.1.1. Optimization of number of potential cycles on the response of LCN2 To investigate the effect of P(ATT)-GO thickness on the current response of LCN2, the different number of potential cycles during the electrodeposition were studied. The P(ATT)-GO was electropolymerized on the GSPE in 0.1 mol L−1 H2SO4 containing 1 mg/mL of GO and 1.0 mmol L−1 ATT using cycles ranging from 2 to 30. As can be seen in Fig. S3, the peak current of LCN2 increased with the increasing number of cycles to 15. The current response slightly decreased with the further increase in the number of potential cycles suggesting that the thicker polymer film could generate a barrier for electron transfer reaction [45]. Thus, the electropolymerization cycles of 15 were chosen to construct the aptasensor in the subsequent experiments. The electrochemical characterization of each modification step was monitored by CV and EIS. Fig. 3(A) shows the CVs of the different modified GPSEs in 0.1 mol L−1 KCl containing 5.0 mmol L−1 Fe (CN)63−/4‒. Bare GSPE exhibited a well-defined redox peak couple (curve a) which corresponds to the reversible redox reaction of Fe (CN)63−/4‒. After the electrodeposition of P(ATT)-GO composite, a remarkable increase in redox peak currents was observed due to the conductive properties of P(ATT)-GO layer (curve b). With the modification of AuNPs onto the GSPE/P(ATT)-GO electrode, the highest redox peak currents are observed which can be ascribed to the good conductivity of the AuNPs (curve c). The GSPE/P(ATT)-GO electrode
3.3. LCN2 determination The voltammetric determination of LCN2 was carried out using the different concentrations of the LCN2. As shown in Fig. 6(A), the current signals of an electroactive product, α-naphthol, increased with increasing concentrations of LCN2 Fig. 6(B). The increase in the peak current with each addition of LCN2 is attributed to the formation of an aptamer-LCN2 complex. The fabricated aptasensor exhibited two linear relationships between the current signal and LCN2 concentration; one from 1.0 ng/mL to 50.0 ng/mL with a determination coefficient (R2) of 0.998 (Fig. 6(C)) and another one from 50.0 ng/mL to 1000 ng/mL with a R2 of 0.981 (Fig. 6(D)). The limit of detection (LOD) and the limit of quantification (LOQ) for LCN2 were calculated according to the following equations and found as 0.3 ng/mL and 1.0 ng/mL, respectively. 4
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Fig. 3. (A) CV and (B) EIS of (a) bare GSPE, (b) GSPE/P(ATT)-GO, (c) GSPE/P(ATT)-GO/AuNPs, (d) GSPE/P(ATT)-GO/AuNPs/Apt1 electrodes in 0.1 mol L−1 KCl solution containing 5.0 mmol L−1 Fe(CN)63−/4‒.
LOD = 3s / m , LOQ = 10s / m
(1)
Table 1 summarizes the results of previously reported LCN2 sensing platforms in terms of the linear ranges and detection limits with our developed aptasensor. As demonstrated above, the fabricated aptasensor exhibited the improvement in the detection limit compared to the other electrodes given in Table 1 [3,36,47]. This might be attributed to the excellent electrical conductivity, high surface area of the fabricated matrix, and good electron communication obtained by the prepared nanocomposite. 3.4. Selectivity and reproducibility To examine the selectivity of the proposed aptasensor for the LCN2 analysis, bovine serum albumin (BSA), human serum albumin (HSA), haptoglobin (HPG), and α-fetoprotein (AFP) as interfering substances were tested under the same conditions. As shown in Fig. 7, the effect caused by the interfering proteins (at the concentration of 500 ng/mL) using GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2 electrode was negligible with the relative standard deviation (RSD) less than 5.0%. Therefore, it can be concluded that the interaction between aptamer molecules and LCN2 is based on specific recognition. Thus, these observations suggest that the sensing mechanism based on the sandwich method is highly specific [48]. Thus, the prepared aptasensor is highly selective for LCN2 determination. The reproducibility of the aptasensor was evaluated by measuring the analytical response of five different electrodes prepared independently under the same conditions. The RSD% results (1.24%) of the DPV peak current signals prove that the aptasensor has good reproducibility.
Fig. 5. The effect of incubation time on the DPV signal of the α-naphthol at the GSPE/P(ATT)-GO/AuNPs. Inset: Current vs. incubation time of LCN2 (n = 3).
3.5. Real sample analysis To further evaluate the performance of the proposed GSPE/P(ATT)GO/Apt1/LCN2/Apt2 electrode, DPV measurements were performed using a fetal bovine serum (FBS). The recovery tests were carried out by standard additions of LCN2 to FBS samples, and the results are in the range from 99.20% and %103.22, and the RSD values were found to be in the range from 1.17% to 4.35% (Table 2). These results obtained for repeatable on each sample indicate the feasibility of the proposed aptasensor for the determination of LCN2 in fetal bovine serum samples. The accuracy of the developed aptasensor was studied via comparison with a commercially available ELISA kit for the LCN2 determination and checked by a t-test. The RSD values were found to be
Fig. 4. The effect of Apt1(A) and Apt2 (B) concentration on the DPV signal of the α-naphthol at the GSPE/P (ATT)-GO/AuNPs. Inset: Current vs. aptamer concentrations (n = 3). 5
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Fig. 6. (A) Differential pulse voltammograms of various concentrations of LCN2. (B) Effect of LCN2 concentration on the voltammetric response of GSPE/P(ATT)GO/AuNPs/Apt1/LCN2/Apt2 electrode. (C), (D) Linear dependence of LCN2 concentration on the α-naphthol signals at GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2 electrode (n = 3). Table 1 Comparison of analytical performances of the different electrochemical sensors for the determination of LCN2. Electrode
Linearity
LOD
Reproducibility
Application
Reference
Aniline functionalized G/PANI nanodroplet-modified electrode LA2/AuNPs/PAMAM-modified gold electrode
50.0–500.0 ng/mL
21.1 ng/mL 1.0 ng/mL
ITO/TiO2/CoPc/CS/SA/BSA/BiNb/NGAL photoelectrochemical sensor GSPE/P(ATT)-GO/AuNPs/Apt1/LCN2/Apt2
1.0–500.0 pg/mL
0.6 pg/mL
RSD: 4.7%
1.0–1000.0 ng/mL
0.3 ng/mL
RSD: 1.24% (n = 5)
Human urine Recoveries:97.5–104.4% Human serum Recoveries:98.5–98.8% Human urine Recoveries:96.1–97.6% Human serum Recoveries: 93.2–112% Fetal bovin serum Recoveries: 99.20–103.22
[3]
50.0–250.0 ng/mL
RSD: 1.49–9.20% (n = 5) RSD: 2.1% (n = 10)
[36]
[47] This study
Graphene (G), polyaniline (PANI), rabbit polygonal lipocalin-2 antibody (LA2), gold nanoparticles (AuNPS), generation-1 polyamidoamine (PAMAM), indium tin oxide (ITO), titanium dioxide (TiO2), streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc), chitosan (CS), streptavidin (SA), Bovine serum albumin (BSA), biotinylated nanobodies (BiNb), neutrophil gelatinase-associated lipocalin (NGAL), relative standard deviation (RSD).
less than 5.0% confirming good accuracy of the proposed sensing strategy (Table 3). The t values of three different LCN2 concentrations were found as 0.96, 0.80 and 0.47 at 95% confidence level for tcritic 2.13. The results obtained from our developed aptasensor are in good agreement with the results obtained from the ELISA method, as exhibited in Table 3.
be prepared via a simple electrodeposition procedure. Surface coverage and electrocatalytic properties of the fabricated GSPE/P(ATT)-GO/ AuNPs were remarkably improved in comparison with unmodified electrode. The developed aptasensor showed high sensitivity, selectivity and low LOD (0.3 ng/mL) value using the DPV technique. Moreover, the feasibility of the sensing strategy was investigated in fetal bovine serum samples using the standard addition method and high recovery values (99.20%–103.22%) were obtained with low RSDs. Importantly, the LCN2 levels measured by DPV technique were compared to a conventional ELISA method and the developed aptasensor presented more effortless preparation procedure, high selectivity and accuracy, rapid analysis, and low-cost sensing with a good correlation. Therefore, this sensing platform could be an efficient tool for the determination of LCN2 levels in clinical research.
4. Conclusion Herein, we prepared a novel aptasensor for the determination of LCN2 based on a sandwich assay. The proposed disposable electrode was easy-to-use, cost-effective and required a low amount of reagents. The modified GSPE was obtained by electrodeposition of graphene oxide-poly-3-amino-1,2,4-triazole-5-thiol composite (GO-P(ATT)) and gold nanoparticles (AuNPs). The characterization of the fabricated aptasensor was carried out by using SEM, XPS, CV and EIS techniques. The prepared electrode has suitable reproducibility and stability and could 6
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[4]
[5] [6] [7]
[8]
[9]
Fig. 7. Selectivity of the proposed aptasensor to 50 ng/mL of LCN2 by comparing it to the various proteins at the 500 ng/mL level.
[10]
Table 2 Results of real sample analysis at different concentrations of LCN2. Sample
Added (ng/mL)
Found (ng/mL)
Recovery%
RSD%a
Fetal bovine serum
5.0 10.0 100.0
4.96 10.18 103.22
99.20 101.80 103.22
2.49 1.17 4.35
a
[11] [12] [13]
Each value is the mean of three measurements. [14]
Table 3 Comparison of real sample analysis results with ELISA method. Sample
Developed aptasensor (ng/mL)
ELISA (ng/ mL)
RSD%a (ELISA)
t
Fetal bovin serum
4.96 10.18 100.22
5.12 9.89 99.47
0.89 2.57 3.42
0.96 0.80 0.47
a
[15] [16] [17]
Each value is the mean of three measurements.
Declaration of competing interest
[18]
We certify that there are no conflicts of interest with any financial organization regarding the material discussed in the manuscript. This study was supported by the scientific and technological research council of Turkey (TUBITAK) under grants 116Z846.
[19]
Acknowledgments
[21]
The authors acknowledge the support provided by the scientific and technological research council of Turkey (TUBITAK) under grants 116Z846.
[22]
[20]
[23] [24]
Appendix A. Supplementary data
[25]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120666.
[26]
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