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Electrocatalytic determination of traces of insulin using a novel silica nanoparticles-Nafion modified glassy carbon electrode
Journal of Electroanalytical Chemistry 714-715 (2014) 70–75
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Electrocatalytic determination of traces of insulin using a novel silica nanoparticles-Nafion modified glassy carbon electrode Nader Amini, Mohammad Bagher Gholivand, Mojtaba Shamsipur ⇑ Department of Chemistry, Razi University, Kermanshah, Iran
a r t i c l e
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Article history: Received 21 July 2013 Received in revised form 21 November 2013 Accepted 12 December 2013 Available online 18 December 2013 Keywords: Silica nanoparticles Nafion, Insulin determination Electrochemical impedance spectroscopy Electrocatalysis
a b s t r a c t A novel electrochemical sensor for the detection of insulin was proposed based on immobilizing silica nanoparticles/Nafion on glassy carbon electrode. Transmission electron microscopy, electrochemical impedance spectroscopy, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to confirm the successful stepwise assembly procedure of the sensor. The electrocatalytical behaviors of the sensor were also investigated by CV and DPV. Results showed that nano-SiO2 exhibited a remarkable electrocatalytic activity for the oxidation of insulin under optimal conditions. The electrocatalytic response of the sensor was proportional to the insulin concentration in the range of 10–50 nM with a limit of detection and sensitivity of 3.1 nM and 300 pAnM1, respectively. The modified electrode show many advantages such as simple preparation without using any special electron transfer mediator or specific reagent, high sensitivity, excellent catalytic activity at physiological pH values, short response time, and remarkable antifouling property toward insulin and its oxidation product. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Insulin is a peptide hormone with one intra chain and two inter chain disulfide bonds and containing a chain of 21 amino acid residues and a B chain of 30 amino acid residues. The determination of the terminal residues of insulin has shown that the sub-molecule with a molecular weight of 12,000 is made up of four open peptide chains bound to gather by disulfide linkages [1,2]. Diabetes, as one of the most prevalent and costly diseases in the world, is caused by the insufficient release of insulin or loss of insulin action at target tissues which results in aberrant glucose and lipid metabolism. The disease is brought about by either too low insulin in the body (type 1 diabetes) or by the bodies that not responding to the effects of insulin (type 2 diabetes) [3–6]. Therefore, detection of insulin is of critical importance in clinical diagnostics. Three main procedures for insulin sensing include bioassay [6–8], immunoassay [9–11] and chromatographic assay [12–14]. However, chromatographic methods require complicated and expensive instrumentations, professional operators, time-consuming detection processes and, usually, complex pre-treatment steps. While, in the case of immunoassay as the most widely used method in this respect [6–8], the precise determination of insulin is hindered by the non-specific binding. Meanwhile, due to its lengthy, relatively imprecise and insensitive procedure, the bioassay is not used by routine clinical laboratories. ⇑ Corresponding author. Tel.: +98 21 66809032; fax: +98 21 66908030. E-mail address: [email protected] (M. Shamsipur). 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.12.015
In the past two decades, an extensive interest has been focused on the design of new electroanalytical methods for the insulin assay. It is well-known that the advantages of electroanalytical techniques including low detection limit, high sensitivity, selectivity towards electroactive species, low cost instruments and wide linear rang provide a valuable way for direct determination of trace amounts of various analytes of biological interest in different samples on a routine basis. Thus, the electrochemical oxidation of insulin has been well established as a basis for monitoring cellular processes [15], application to flow systems [16] and using as sensitive the amperometric detectors [17] for this important protein. However, the electrochemical detection of insulin is restricted by the slow kinetics of insulin oxidation on the ordinary electrode materials such as glassy carbon. As a result, a variety of chemically modified electrodes has been suggested for promoting the oxidation and detection of insulin. In 1989, Cox and Gray reported the use of a ruthenium dioxide-cyanoruthenate film for acceleration of insulin oxidation in acidic media [18]. Since then, a number of reports for the insulin determination based on the electrode modifications with ruthenium oxide [19], cobalt oxide nanostructure [20], Ru(II) metallodendrimer [15,21], iridium oxide [17], carbon nanotubes [22,23], RuOx/carbon nanotubes [24], nickel nanoparticles [25], guanine/nickel oxide nanoparticles [26] sol–gel derived carbon ceramic composite [1], nickel oxide nanoparticles-multiwalled carbon nanotubes [27], silica gel modified carbon paste electrode [28], silicon carbide nanoparticles [29], nanocarbon black electrode surface [30], cobalt oxide nanostructure [31], carbon nanotube–nickel–cobaltoxide modified electrode [32] and carbon
N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75
nanotube [33] have appeared in the literature. Although the modified electrodes have been successfully employed for monitoring of insulin, some of them possess such disadvantages as reduced stability under physiological condition, high detection limit, poor long term stability and complicated multi-step preparation procedures. Silica is a common name for materials composed of silicon dioxide (SiO2) occurring in crystalline and amorphous forms. The application of synthetic amorphous silica, especially its nanoparticles, has received wide attention in a variety of industries [34]. Silica nanoparticles have also found an extensive application in chemical mechanical polishing and as additives to drugs, cosmetics, printer toners, varnishes and foods. In recent years, the use of SiO2 nanoparticles (NPs) has been extended to biochemical and biotechnological fields, such as biosensors for simultaneous assay of glucose, lactate, L-glutamate and hypoxanthine levels in rat striatum [35], biomarkers for leukemia cell identification using optical microscopy imaging [36], cancer therapy [37], DNA delivery [38], drug delivery [39] and enzyme immobilization [40]. SiO2 nanoparticles are also excellent matrices for enzyme immobilization due to their good biocompatibility and easy preparation. Hu et al. immobilized several hemeproteins with SiO2 nanoparticles through the layer-by-layer assembly [41], and investigated the driving forces for the assembly procedure [42]. In addition, in some circumstances, the oxide nanoparticles can be used as labels for biomolecules. Fang and co-workers have reported the application of tris(2,20 -bipyridyl)cobalt(II) doped SiO2 nanoparticles as oligonucleotide labels for electrochemical detection of DNA hybridization [43]. Thus, based on the above mentioned examples, SiO2NPs seem to be excellent candidates for construction of novel and improved sensing devices and, in particular, electrochemical sensors and biosensors. In this work, we wish to report the first application of SiO2NPs as suitable electrocatalyst for the oxidation of insulin at physiological pH. Although SiO2NPs possess well-defined electron transfer properties on the surfaces of glassy carbon electrode (GCE), the films are easily washed from the electrode with buffer solution and become unstable during the potential scan. To overcome this problem, we immobilized SiO2NPs at GCE by using Nafion polymer (Nf). Thus, when SiO2NPs were conjugated with Nf of favorable compatibility and film-forming ability, a promising platform for electrochemical analysis of insulin based on SiO2NPs/Nf was developed. Due to stability and antifouling properties of SiO2NPs, the prepared silica nanoparticles/nafion modified glassy carbon electrode was then used as a highly efficient amperometric sensor for the detection of insulin in nanomolar scale.
[KCl (sat)] reference electrode, a Pt wire counter electrode and a SiO2 nanoparticles modified glassy carbon working electrode was used. All electrodes were purchased from Metrohm. A personal computer was used for data storage and processing. 2.3. Preparation of the modified electrode Prior to the modification, the bare GCE was polished successively with Nos. 1–6 emery papers and 0.5 lm alumina slurry to a mirror-like smoothness surface. A 1.0 v/v% Nafion solution was prepared by diluting the 5.0 v/v% Nafion solution in ethanol. 3 mg of silica nanoparticles was added to 5 mL 1.0 v/v% Nafion solution and ultrasonicated for 30 min to form a homogeneous SiO2NPs-Nafion solution. Then, 5 lL of above solution was casted onto cleaned GC electrode surface and dried in air at room temperature. 3. Results and discussion 3.1. Morphological and electrochemical characterization of SiO2NPsNafion/GCE The TEM image of prepared SiO2NPs-Nafion/GCE is shown in Fig. 1, confirming the successful attachment of silica nanoparticles directly on the glassy carbon electrode. As seen, uniformly distributed silica nanoparticles with an average diameter ranging from 30 to 40 nm are present at the surface of electrode. Electrochemical impedance spectroscopy (EIS) is well known for providing information about the interface characters of the electrode surface during the modification process [44,45]. Thus, the Nyquist plots for different modified electrodes were obtained by using 5 mM of [Fe(CN)6 ]3-/4- redox couples, as the electrochemical probe, and the results are shown in Fig. 2. The semicircle portions observed in EIS corresponded to the electron-transfer-limited processes at the surface of electrodes. Fig. 2 shows the results of EIS on bare GCE (a) and modified SiO2NPs-Nafion/GCE (b) in the presence of [Fe(CN)6 ]3-/4- redox probe measured at the corresponding formal potentials. EIS of the bare GCE (Fig. 2a) exhibited an almost straight line, which implied the characteristic of a diffuse limiting
2. Experimental 2.1. Reagents Bovine insulin (5800, >27 USP units mg1) was from Sigma. Silica nanoparticle was purchased from Sigma–Aldrich. Nafio (5.0 v/v%) was obtained from Sigma Chemical Co. Buffer solutions (0.1 M) were prepared from disodium hydrogen phosphate (Na2HPO4), sodium di-hydrogen phosphate (NaH2PO4), hydrochloric acid (HCl) and sodium hydroxide (NaOH), all from Merck. All electrochemical experiments were carried out at 25.0 ± 0.1 °C. 2.2. Apparatus Electrochemical experiments were performed on a computer controlled l-Autolab modular electrochemical system (Eco Chemie U/techt, the Netherlands), driven with GPES software (Eco Chemie). A conventional three-electrode cell consisting of an Ag/AgCl
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Fig. 1. TEM image of SiO2NPs-Nafion/GCE.
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N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75
Fig. 2. Electrochemical impedance spectroscopy responses of bare GCE (a) and SiO2NPs-Nafion/GCE in 5 mM [Fe(CN)6 ]3-/4- redox couple.
step of the electrochemical processes. After modification of the GCE with SiO2NPs-Nafion, an obvious interfacial Rct was observed (Fig. 2b), indicating the formation of an insulating SiO2NPs-Nafion layer at the electron conductor of the redox probe. However, it should be noted that the presence of a repulsion force between negatively charged Nafion and Fe(CN)63/4- anion is also an important factor in the increased resistance at the modified electrode.
3.2. Electrocatalytic oxidation of insulin on SiO2NPs-Nafion/GCE
Fig. 3. Cyclic voltammetry responses of the bare GCE (a and b) and modified SiO2NPs-Nafion/GCE electrodes (c and d) in the absence (a and c) and presence 50 lM of insulin (b and d) at a scan rate of 30 mV s1 and a pH of 7.35.
The electrocatalytic activity of SiO2-Nafion modified GC electrode was investigated by cyclic voltammetry. Fig. 3 shows the cyclic voltammograms of modified and unmodified electrodes in the absence and presence of 50 lM insulin in a 0.1 M buffer solution of pH 7.35. As seen, for the bare GCE, no redox response of insulin can be seen in the potential range 0.4–0.85 V. However, at the modified SiO2NPs-Nafion/GCE, the oxidation current greatly increased due to catalytic oxidation of insulin. A substantial negative shift of the anodic peak potential and dramatic increase of current indicated excellent catalytic ability of SiO2NPs towards insulin oxidation. It should be noted that, at the surface of SiO2NPs modified GCE, not only the overvoltage for insulin oxidation decreased significantly but also antifouling properties of modified film improved the reproducibility of the electrode system. Therefore, SiO2NPs seems to act as suitable mediators to shuttle electron between insulin and working electrode and facilitate the electrochemical regeneration following the electron exchange with insulin. Based on some previously published documents [46–48], a possible reaction mechanism between Nafion, SiOH (prepared from SiO2NPs in ethanolic Nafion solution) and thio-amino acids of insulin can be presented as:
with the sulfonic acid group of Nafion. The resulting SiOH can consequently abstract a proton from thio-amino acids of insulin. The strong electrostatic interaction between the resulting [Si-OH2]+/ insulin thio-amino acid-S- pair at the solution-electrode surface interface can then facilitate the oxidation of insulin at the prepared SiO2NPs-Nafion/GCE modified electrode. The best loading of the silica nanoparticles on the GC electrode surface was found to be casting of 5 lL of a 5 mL 1.0 v/v% Nafion ethanol solution containing 3 mg of silica nanoparticles, as described in Section 2.3. At lower SiO2NPs loadings, the proper oxidation of insulin could not be achieved at the surface of electrode, while, the higher loadings of the modifier acted as an insulator of the electrode surface. In order to optimize the electrocatalytic response of modified electrodes toward insulin oxidation, the influence of solution pH on the catalytic oxidation behavior of insulin at the surface of the modified electrode was investigated. The CVs of a 50 lM solution of insulin at different pH values (from 3 to 11) at the SiO2NPs modified GC electrode were recorded (Fig. 4). As seen, maximum peak current and most reproducible results were achieved at a pH of 7.35. While, at lower and higher pH values, the voltammetric signal diminished significantly. Thus, the insulin measurements were carried out at a physiological pH of 7.35 in further studies.
As is obvious, the reaction of water with Si(OC2H5)4 (prepared from SiO2NPs and ethanol present in Nafion solution) will result in the preparation Si(OH), which is stabilized via hydrogen bonding
In order to evaluate the electrocatalytic activity of SiO2NPs, the cyclic voltammograms of the modified electrode in the presence of varying concentrations of insulin were recorded and the results are
N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75
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Fig. 4. The CVs of a 50 lM solution of insulin at different pH values (from 7 to 9) at the SiO2NPs modified GCE.
shown in Fig. 5. As seen, the insulin anodic peak current increased with increasing insulin concentration in solution. The resulting linear graph of oxidation peak current against insulin concentration (inset of Fig. 4) possessed a regression equation of I (lA) = 164,333 [insulin] lA lM1 + 12,156 lA, R2 = 0.9965. The limit of detection was estimated as 30 nM at a signal to noise of 3. From the results thus obtained, it can be inferred that the presence of the posited SiO2NPs at the surface of GC electrode facilitates the detection of insulin at low concentration levels. The cyclic voltammograms of 50 lM insulin in a 0.1 M buffer solution of pH 7.35 at different scan rates were recorded (Fig. 6). As it is seen from the inset of Fig. 5, the peak current for oxidation of insulin is proportional to square root of the scan rate, suggesting that the process is controlled by diffusion as expected for a catalytic system [49]. The differential pulse voltammograms of the modified electrode designed in 0.1 M buffer solutions of pH 7.35 containing different concentration of insulin, from 10 to 50 nM, were recorded and the results are shown in Fig. 7. As can be seen from the inset of
Fig. 6. Cyclic voltammetry responses SiO2NPs-Nafion/GCE in a 0.1 M phosphate buffer solution of pH 7.35 containing 0.1 lM of insulin at different scan rates: (a) 10, (b) 20, (c) 30, (d) 40, (e) 50, (f) 60, (g) 70, (h) 80, (i) 90 and (j) 100 mV s1. Inset shows a linear plot of peak current vs. m1/2.
Fig. 7, there is a nice linear relationship between the catalytic currents vs concentration of insulin, which can be fitted to the regression equation DI = (Iobs Ibg) (nA) = 0.3 [insulin] nA nM1–2.5 nA, with R2 = 0.9885. The limit of detection, evaluated at a signal to noise ratio of 3, was found to be 3.1 nM. 3.3. Interference study Possible interference for the detection of insulin at SiO2NPsNafion/GCE was investigated by addition of several potential interferences including glucose, glycine, phenylalanine, histidine, cystine and tryptophan to a 0.1 M buffer solution of pH 7.35 in the presence of 30 lg mL1 of LEV. Except with tryptophan, in all other cases a 50-fold excess concentration of the common compounds used did not show any measurable interference in insulin detection. However, in the case of some excess amount of tryptophan, the electrode’s response currents showed a measurable increase. 3.4. Repeatability and stability
Fig. 5. Cyclic voltammograms of SiO2NPs-Nafion/GCE in a 0.1 M buffer solutions of pH 7.35 containing varying concentrations of insulin: (a) 0.0, (b) 0.1, (c) 0.2, (d) 0.3, (e) 0.4, (f) 0.5, (g) 0.6, (h) 0.7 and (i) 0.8 lM. Inset shows a linear plot of peak current vs. insulin concentration.
To evaluate the precision of the proposed sensor, the electrochemical experiments were repeatedly performed for six successive assays at an insulin concentration of 0.8 lM using the SiO2NPs-Nafion/GCE. The relative standard deviation was found to be 4.0%, which revealed an excellent repeatability for the electrochemical system designed. The stability and lifetime the modified electrode was also investigated. After the same SiO2NPs-Nafion/GCE was used for several times during one and four weeks, the electrode response was
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N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75 Table 2 Determination of insulin in a Bovine insulin injection. Insulin added (lM)
b
0.0 0.4 0.6 0.8 a b
Insulin found (lM)a 0.207 ± 0.011 0.389 ± 0.084 0.609 ± 0.019 0.817 ± 0.011
Recovery (%) 103.5 97.2 101.1 102.1
Average of four determinations ± relative standard deviation. The expected concentration of insulin claimed by the producer was 0.2 lM.
3.5. Comparison of the figures of merit of the proposed sensor with those of previous electrochemical methods
Fig. 7. Differential pulse voltammograms SiO2NPs-Nafion/GCE in a 0.1 M phosphate buffer solution of pH 7.35 containing different concentration of insulin: (a) 0, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50 nM. Inset shows a linear plot of peak current vs. insulin concentration.
Table 1 compares the linear concentration range, limit of detection, sensitivity and optimized pH of the proposed sensor for insulin determination with those of other insulin electrodes previously reported in the literature [14,15,22–24,39–42]. As it is obvious from Table 1, except with the case of NiNPs/CCE [24], Si/CPE [28], CoO/GCE [31], CNT/Ni/CoO/CNT [32] and CNT [33] sensors, the LOD of the proposed SiO2NPs-Nafion/GCE is superior to the other reported sensor for detection of insulin. However, three of these electrodes work beyond the biological pH, at highly acidic and basic pHs [24,28,31], and most of them have not been applied to insulin determination in real biological samples such as skin sweat, which was tested in this work. Moreover, the proposed electrochemical sensor possesses a short response time, low cost and could be used repeatedly at biological pH for more than a month without any significant divergence in its response. 3.6. Analytical applications
found to gradually decrease to 96% and 89% of the initial current, respectively. Such a small decrease of current sensitivity of about 4–11% can be attributed to the high stability of the modified electrode designed.
In order to check its practical application, the proposed sensor was used to determine insulin in its commercial injection (Table 2) and in a skin sweat sample (Table 3) using the recommended procedure. In addition, a certain value of standard solution of
Table 1 Analytical parameters of several modified electrodes for determination of insulin. Electrode
Method
pH
LOD (nM)
Sensitivity (nA/lM)
Dynamic linear range (nM)
Refs.
IrO/GCEa RuRDMs/CEb RuOx/CPEc CNTe/GCE Chitosan-CNT/GCE NiNPs/CCEf
Amp FIA FIAd FIA Amp Amp FIA Amp Amp DPV DPV Amp Amp DPV Amp FIA Microfluidic FIA CV DPV
7.4 7 7.4 7.4 7.4 13
20 2 50 14 30 0.04 0.0026 5 0.036 20 5 0.01 0.22 0.001 500 23 <1000 – 30 3.1
35.2 2.3 102 0.875 48 1.35 102 4 103 2.7 105 – 0.107 864 – 83.9 27.57 0.001 0.441 0.072 1.33 2.4 106 164,333 20
50–500 6–400 100–1000 100–1000 100–3000 0.1–700 0.015–0.1 20–260 0.09–1.4 100–1000 20–1000 0.1–15 0.1–31.5 3.4–68 50–500 100–1000 100–10,000 1.9 105-1.8 106 100–800 10–50
[14] [15] [20] [22] [23] [24]
NiO/MWCNT/SPE Si/CPE SiC/GCE ABNCg/CPE CoO/GCE CNT/Ni/CoO/CNT CNT RuO/RuCN/CFMEh RuOx/CFME MWCNT/DHPi Ru-inorganic polymer film/GCE SiO2NPs-Nafion/GCE a b c d e f g h i
10 2 7.4 10 9 7.5 7.4 7.4 7.4 7.4 2 7.35
Glassy carbon electrode. Ruthenium metallodendrimer multilayers/carbon electrode. Carbon paste electrode. Flow injection analysis. Carbon nanotubes. Carbon ceramic electrode. Acetylene black nanocarbon. Carbon fiber microelectrode. Multi-wall carbon nanotube/Dihydropyran.
[27] [28] [29] [30] [31] [32] [33] [39] [40] [41] [42] This work
N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75 Table 3 Determination of insulin in a skin sweat sample. Insulin added (lM) 0.0 0.4 0.6 a b
Insulin found (lM)a 0.218 ± 0.013 0.383 ± 0.036 0.616 ± 0.032
b
Recovery (%) 109.5 95.0 102.0
Average of four determinations ± relative standard deviation. The expected concentration of insulin claimed by the producer was 0.2 lM.
insulin was added into the corresponding samples for testing the recovery. The precision and excellent recoveries reported in Tables 2 and 3 clearly indicated the reliability of the proposed SiO2NPs-Nafion/GCE electrochemical sensor for the insulin determination in real samples of insulin. 4. Conclusion In conclusion, we reported a novel electrochemical sensing strategy for sensitive detection of insulin. The electrooxidation of nanomolar insulin solution was successfully preformed using the GCE modified with SiO2 nanoparticles. The modified electrode shows excellent electrocatalytic activity, antifouling, significantly lower detection limit, grater analytical sensitivity, simple preparation and stability response. The response characteristics of the proposed electrochemical sensor are significantly improved compared to most of some modified electrodes reported as insulin sensors. The results suggested that it is feasible to apply the proposed method to quantitative determination of insulin at physiological pH. Acknowledgments This research was supported by the Iranian Nanotechnology Initiative and Razi University Research Council. References [1] A. Salimi, S. Pourbeyram, H. Hadadzadeh, J. Electroanal. Chem. 542 (2003) 39– 49. [2] A. Arvinte, A.C. Westermann, A. Mariesesay, V. Virtanen, Sens. Actuators, B 150 (2010) 756–763. [3] S.A. Ross, E.A. Gulve, M. Wang, Chem. Rev. 104 (2004) 1255–1282. [4] B.J. Parakasam, S.K. Vareed, L.K. Olson, M.G. Nair, J. Agric. Food. Chem. 53 (2005) 28–31. [5] National Diabetes Fact Sheets: General and National Estimates on Diabetes in the United States, Centers for Disease Control and prevention, 2000. [6] D. Keller, R. Clausen, K. Josefen, J.J. Led, Biochemistry 40 (2001) 10732–10740. [7] C.P. Reis, A.J. Ribeiro, S. Houng, F. Veiga, J. Neufeld, Eur. J. Pharmaceut. Sci. 30 (2007) 392–397. [8] R. Sreekanth, V. Pattabhi, S.S. Rajan, Biochimica 90 (2008) 467–473.
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Electrocatalytic determination of traces of insulin using a novel silica nanoparticles-Nafion modified glassy carbon electrode Nader Amini, Mohammad Bagher Gholivand, Mojtaba Shamsipur ⇑ Department of Chemistry, Razi University, Kermanshah, Iran
a r t i c l e
i n f o
Article history: Received 21 July 2013 Received in revised form 21 November 2013 Accepted 12 December 2013 Available online 18 December 2013 Keywords: Silica nanoparticles Nafion, Insulin determination Electrochemical impedance spectroscopy Electrocatalysis
a b s t r a c t A novel electrochemical sensor for the detection of insulin was proposed based on immobilizing silica nanoparticles/Nafion on glassy carbon electrode. Transmission electron microscopy, electrochemical impedance spectroscopy, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to confirm the successful stepwise assembly procedure of the sensor. The electrocatalytical behaviors of the sensor were also investigated by CV and DPV. Results showed that nano-SiO2 exhibited a remarkable electrocatalytic activity for the oxidation of insulin under optimal conditions. The electrocatalytic response of the sensor was proportional to the insulin concentration in the range of 10–50 nM with a limit of detection and sensitivity of 3.1 nM and 300 pAnM1, respectively. The modified electrode show many advantages such as simple preparation without using any special electron transfer mediator or specific reagent, high sensitivity, excellent catalytic activity at physiological pH values, short response time, and remarkable antifouling property toward insulin and its oxidation product. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Insulin is a peptide hormone with one intra chain and two inter chain disulfide bonds and containing a chain of 21 amino acid residues and a B chain of 30 amino acid residues. The determination of the terminal residues of insulin has shown that the sub-molecule with a molecular weight of 12,000 is made up of four open peptide chains bound to gather by disulfide linkages [1,2]. Diabetes, as one of the most prevalent and costly diseases in the world, is caused by the insufficient release of insulin or loss of insulin action at target tissues which results in aberrant glucose and lipid metabolism. The disease is brought about by either too low insulin in the body (type 1 diabetes) or by the bodies that not responding to the effects of insulin (type 2 diabetes) [3–6]. Therefore, detection of insulin is of critical importance in clinical diagnostics. Three main procedures for insulin sensing include bioassay [6–8], immunoassay [9–11] and chromatographic assay [12–14]. However, chromatographic methods require complicated and expensive instrumentations, professional operators, time-consuming detection processes and, usually, complex pre-treatment steps. While, in the case of immunoassay as the most widely used method in this respect [6–8], the precise determination of insulin is hindered by the non-specific binding. Meanwhile, due to its lengthy, relatively imprecise and insensitive procedure, the bioassay is not used by routine clinical laboratories. ⇑ Corresponding author. Tel.: +98 21 66809032; fax: +98 21 66908030. E-mail address: [email protected] (M. Shamsipur). 1572-6657/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2013.12.015
In the past two decades, an extensive interest has been focused on the design of new electroanalytical methods for the insulin assay. It is well-known that the advantages of electroanalytical techniques including low detection limit, high sensitivity, selectivity towards electroactive species, low cost instruments and wide linear rang provide a valuable way for direct determination of trace amounts of various analytes of biological interest in different samples on a routine basis. Thus, the electrochemical oxidation of insulin has been well established as a basis for monitoring cellular processes [15], application to flow systems [16] and using as sensitive the amperometric detectors [17] for this important protein. However, the electrochemical detection of insulin is restricted by the slow kinetics of insulin oxidation on the ordinary electrode materials such as glassy carbon. As a result, a variety of chemically modified electrodes has been suggested for promoting the oxidation and detection of insulin. In 1989, Cox and Gray reported the use of a ruthenium dioxide-cyanoruthenate film for acceleration of insulin oxidation in acidic media [18]. Since then, a number of reports for the insulin determination based on the electrode modifications with ruthenium oxide [19], cobalt oxide nanostructure [20], Ru(II) metallodendrimer [15,21], iridium oxide [17], carbon nanotubes [22,23], RuOx/carbon nanotubes [24], nickel nanoparticles [25], guanine/nickel oxide nanoparticles [26] sol–gel derived carbon ceramic composite [1], nickel oxide nanoparticles-multiwalled carbon nanotubes [27], silica gel modified carbon paste electrode [28], silicon carbide nanoparticles [29], nanocarbon black electrode surface [30], cobalt oxide nanostructure [31], carbon nanotube–nickel–cobaltoxide modified electrode [32] and carbon
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nanotube [33] have appeared in the literature. Although the modified electrodes have been successfully employed for monitoring of insulin, some of them possess such disadvantages as reduced stability under physiological condition, high detection limit, poor long term stability and complicated multi-step preparation procedures. Silica is a common name for materials composed of silicon dioxide (SiO2) occurring in crystalline and amorphous forms. The application of synthetic amorphous silica, especially its nanoparticles, has received wide attention in a variety of industries [34]. Silica nanoparticles have also found an extensive application in chemical mechanical polishing and as additives to drugs, cosmetics, printer toners, varnishes and foods. In recent years, the use of SiO2 nanoparticles (NPs) has been extended to biochemical and biotechnological fields, such as biosensors for simultaneous assay of glucose, lactate, L-glutamate and hypoxanthine levels in rat striatum [35], biomarkers for leukemia cell identification using optical microscopy imaging [36], cancer therapy [37], DNA delivery [38], drug delivery [39] and enzyme immobilization [40]. SiO2 nanoparticles are also excellent matrices for enzyme immobilization due to their good biocompatibility and easy preparation. Hu et al. immobilized several hemeproteins with SiO2 nanoparticles through the layer-by-layer assembly [41], and investigated the driving forces for the assembly procedure [42]. In addition, in some circumstances, the oxide nanoparticles can be used as labels for biomolecules. Fang and co-workers have reported the application of tris(2,20 -bipyridyl)cobalt(II) doped SiO2 nanoparticles as oligonucleotide labels for electrochemical detection of DNA hybridization [43]. Thus, based on the above mentioned examples, SiO2NPs seem to be excellent candidates for construction of novel and improved sensing devices and, in particular, electrochemical sensors and biosensors. In this work, we wish to report the first application of SiO2NPs as suitable electrocatalyst for the oxidation of insulin at physiological pH. Although SiO2NPs possess well-defined electron transfer properties on the surfaces of glassy carbon electrode (GCE), the films are easily washed from the electrode with buffer solution and become unstable during the potential scan. To overcome this problem, we immobilized SiO2NPs at GCE by using Nafion polymer (Nf). Thus, when SiO2NPs were conjugated with Nf of favorable compatibility and film-forming ability, a promising platform for electrochemical analysis of insulin based on SiO2NPs/Nf was developed. Due to stability and antifouling properties of SiO2NPs, the prepared silica nanoparticles/nafion modified glassy carbon electrode was then used as a highly efficient amperometric sensor for the detection of insulin in nanomolar scale.
[KCl (sat)] reference electrode, a Pt wire counter electrode and a SiO2 nanoparticles modified glassy carbon working electrode was used. All electrodes were purchased from Metrohm. A personal computer was used for data storage and processing. 2.3. Preparation of the modified electrode Prior to the modification, the bare GCE was polished successively with Nos. 1–6 emery papers and 0.5 lm alumina slurry to a mirror-like smoothness surface. A 1.0 v/v% Nafion solution was prepared by diluting the 5.0 v/v% Nafion solution in ethanol. 3 mg of silica nanoparticles was added to 5 mL 1.0 v/v% Nafion solution and ultrasonicated for 30 min to form a homogeneous SiO2NPs-Nafion solution. Then, 5 lL of above solution was casted onto cleaned GC electrode surface and dried in air at room temperature. 3. Results and discussion 3.1. Morphological and electrochemical characterization of SiO2NPsNafion/GCE The TEM image of prepared SiO2NPs-Nafion/GCE is shown in Fig. 1, confirming the successful attachment of silica nanoparticles directly on the glassy carbon electrode. As seen, uniformly distributed silica nanoparticles with an average diameter ranging from 30 to 40 nm are present at the surface of electrode. Electrochemical impedance spectroscopy (EIS) is well known for providing information about the interface characters of the electrode surface during the modification process [44,45]. Thus, the Nyquist plots for different modified electrodes were obtained by using 5 mM of [Fe(CN)6 ]3-/4- redox couples, as the electrochemical probe, and the results are shown in Fig. 2. The semicircle portions observed in EIS corresponded to the electron-transfer-limited processes at the surface of electrodes. Fig. 2 shows the results of EIS on bare GCE (a) and modified SiO2NPs-Nafion/GCE (b) in the presence of [Fe(CN)6 ]3-/4- redox probe measured at the corresponding formal potentials. EIS of the bare GCE (Fig. 2a) exhibited an almost straight line, which implied the characteristic of a diffuse limiting
2. Experimental 2.1. Reagents Bovine insulin (5800, >27 USP units mg1) was from Sigma. Silica nanoparticle was purchased from Sigma–Aldrich. Nafio (5.0 v/v%) was obtained from Sigma Chemical Co. Buffer solutions (0.1 M) were prepared from disodium hydrogen phosphate (Na2HPO4), sodium di-hydrogen phosphate (NaH2PO4), hydrochloric acid (HCl) and sodium hydroxide (NaOH), all from Merck. All electrochemical experiments were carried out at 25.0 ± 0.1 °C. 2.2. Apparatus Electrochemical experiments were performed on a computer controlled l-Autolab modular electrochemical system (Eco Chemie U/techt, the Netherlands), driven with GPES software (Eco Chemie). A conventional three-electrode cell consisting of an Ag/AgCl
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Fig. 1. TEM image of SiO2NPs-Nafion/GCE.
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Fig. 2. Electrochemical impedance spectroscopy responses of bare GCE (a) and SiO2NPs-Nafion/GCE in 5 mM [Fe(CN)6 ]3-/4- redox couple.
step of the electrochemical processes. After modification of the GCE with SiO2NPs-Nafion, an obvious interfacial Rct was observed (Fig. 2b), indicating the formation of an insulating SiO2NPs-Nafion layer at the electron conductor of the redox probe. However, it should be noted that the presence of a repulsion force between negatively charged Nafion and Fe(CN)63/4- anion is also an important factor in the increased resistance at the modified electrode.
3.2. Electrocatalytic oxidation of insulin on SiO2NPs-Nafion/GCE
Fig. 3. Cyclic voltammetry responses of the bare GCE (a and b) and modified SiO2NPs-Nafion/GCE electrodes (c and d) in the absence (a and c) and presence 50 lM of insulin (b and d) at a scan rate of 30 mV s1 and a pH of 7.35.
The electrocatalytic activity of SiO2-Nafion modified GC electrode was investigated by cyclic voltammetry. Fig. 3 shows the cyclic voltammograms of modified and unmodified electrodes in the absence and presence of 50 lM insulin in a 0.1 M buffer solution of pH 7.35. As seen, for the bare GCE, no redox response of insulin can be seen in the potential range 0.4–0.85 V. However, at the modified SiO2NPs-Nafion/GCE, the oxidation current greatly increased due to catalytic oxidation of insulin. A substantial negative shift of the anodic peak potential and dramatic increase of current indicated excellent catalytic ability of SiO2NPs towards insulin oxidation. It should be noted that, at the surface of SiO2NPs modified GCE, not only the overvoltage for insulin oxidation decreased significantly but also antifouling properties of modified film improved the reproducibility of the electrode system. Therefore, SiO2NPs seems to act as suitable mediators to shuttle electron between insulin and working electrode and facilitate the electrochemical regeneration following the electron exchange with insulin. Based on some previously published documents [46–48], a possible reaction mechanism between Nafion, SiOH (prepared from SiO2NPs in ethanolic Nafion solution) and thio-amino acids of insulin can be presented as:
with the sulfonic acid group of Nafion. The resulting SiOH can consequently abstract a proton from thio-amino acids of insulin. The strong electrostatic interaction between the resulting [Si-OH2]+/ insulin thio-amino acid-S- pair at the solution-electrode surface interface can then facilitate the oxidation of insulin at the prepared SiO2NPs-Nafion/GCE modified electrode. The best loading of the silica nanoparticles on the GC electrode surface was found to be casting of 5 lL of a 5 mL 1.0 v/v% Nafion ethanol solution containing 3 mg of silica nanoparticles, as described in Section 2.3. At lower SiO2NPs loadings, the proper oxidation of insulin could not be achieved at the surface of electrode, while, the higher loadings of the modifier acted as an insulator of the electrode surface. In order to optimize the electrocatalytic response of modified electrodes toward insulin oxidation, the influence of solution pH on the catalytic oxidation behavior of insulin at the surface of the modified electrode was investigated. The CVs of a 50 lM solution of insulin at different pH values (from 3 to 11) at the SiO2NPs modified GC electrode were recorded (Fig. 4). As seen, maximum peak current and most reproducible results were achieved at a pH of 7.35. While, at lower and higher pH values, the voltammetric signal diminished significantly. Thus, the insulin measurements were carried out at a physiological pH of 7.35 in further studies.
As is obvious, the reaction of water with Si(OC2H5)4 (prepared from SiO2NPs and ethanol present in Nafion solution) will result in the preparation Si(OH), which is stabilized via hydrogen bonding
In order to evaluate the electrocatalytic activity of SiO2NPs, the cyclic voltammograms of the modified electrode in the presence of varying concentrations of insulin were recorded and the results are
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Fig. 4. The CVs of a 50 lM solution of insulin at different pH values (from 7 to 9) at the SiO2NPs modified GCE.
shown in Fig. 5. As seen, the insulin anodic peak current increased with increasing insulin concentration in solution. The resulting linear graph of oxidation peak current against insulin concentration (inset of Fig. 4) possessed a regression equation of I (lA) = 164,333 [insulin] lA lM1 + 12,156 lA, R2 = 0.9965. The limit of detection was estimated as 30 nM at a signal to noise of 3. From the results thus obtained, it can be inferred that the presence of the posited SiO2NPs at the surface of GC electrode facilitates the detection of insulin at low concentration levels. The cyclic voltammograms of 50 lM insulin in a 0.1 M buffer solution of pH 7.35 at different scan rates were recorded (Fig. 6). As it is seen from the inset of Fig. 5, the peak current for oxidation of insulin is proportional to square root of the scan rate, suggesting that the process is controlled by diffusion as expected for a catalytic system [49]. The differential pulse voltammograms of the modified electrode designed in 0.1 M buffer solutions of pH 7.35 containing different concentration of insulin, from 10 to 50 nM, were recorded and the results are shown in Fig. 7. As can be seen from the inset of
Fig. 6. Cyclic voltammetry responses SiO2NPs-Nafion/GCE in a 0.1 M phosphate buffer solution of pH 7.35 containing 0.1 lM of insulin at different scan rates: (a) 10, (b) 20, (c) 30, (d) 40, (e) 50, (f) 60, (g) 70, (h) 80, (i) 90 and (j) 100 mV s1. Inset shows a linear plot of peak current vs. m1/2.
Fig. 7, there is a nice linear relationship between the catalytic currents vs concentration of insulin, which can be fitted to the regression equation DI = (Iobs Ibg) (nA) = 0.3 [insulin] nA nM1–2.5 nA, with R2 = 0.9885. The limit of detection, evaluated at a signal to noise ratio of 3, was found to be 3.1 nM. 3.3. Interference study Possible interference for the detection of insulin at SiO2NPsNafion/GCE was investigated by addition of several potential interferences including glucose, glycine, phenylalanine, histidine, cystine and tryptophan to a 0.1 M buffer solution of pH 7.35 in the presence of 30 lg mL1 of LEV. Except with tryptophan, in all other cases a 50-fold excess concentration of the common compounds used did not show any measurable interference in insulin detection. However, in the case of some excess amount of tryptophan, the electrode’s response currents showed a measurable increase. 3.4. Repeatability and stability
Fig. 5. Cyclic voltammograms of SiO2NPs-Nafion/GCE in a 0.1 M buffer solutions of pH 7.35 containing varying concentrations of insulin: (a) 0.0, (b) 0.1, (c) 0.2, (d) 0.3, (e) 0.4, (f) 0.5, (g) 0.6, (h) 0.7 and (i) 0.8 lM. Inset shows a linear plot of peak current vs. insulin concentration.
To evaluate the precision of the proposed sensor, the electrochemical experiments were repeatedly performed for six successive assays at an insulin concentration of 0.8 lM using the SiO2NPs-Nafion/GCE. The relative standard deviation was found to be 4.0%, which revealed an excellent repeatability for the electrochemical system designed. The stability and lifetime the modified electrode was also investigated. After the same SiO2NPs-Nafion/GCE was used for several times during one and four weeks, the electrode response was
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N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75 Table 2 Determination of insulin in a Bovine insulin injection. Insulin added (lM)
b
0.0 0.4 0.6 0.8 a b
Insulin found (lM)a 0.207 ± 0.011 0.389 ± 0.084 0.609 ± 0.019 0.817 ± 0.011
Recovery (%) 103.5 97.2 101.1 102.1
Average of four determinations ± relative standard deviation. The expected concentration of insulin claimed by the producer was 0.2 lM.
3.5. Comparison of the figures of merit of the proposed sensor with those of previous electrochemical methods
Fig. 7. Differential pulse voltammograms SiO2NPs-Nafion/GCE in a 0.1 M phosphate buffer solution of pH 7.35 containing different concentration of insulin: (a) 0, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50 nM. Inset shows a linear plot of peak current vs. insulin concentration.
Table 1 compares the linear concentration range, limit of detection, sensitivity and optimized pH of the proposed sensor for insulin determination with those of other insulin electrodes previously reported in the literature [14,15,22–24,39–42]. As it is obvious from Table 1, except with the case of NiNPs/CCE [24], Si/CPE [28], CoO/GCE [31], CNT/Ni/CoO/CNT [32] and CNT [33] sensors, the LOD of the proposed SiO2NPs-Nafion/GCE is superior to the other reported sensor for detection of insulin. However, three of these electrodes work beyond the biological pH, at highly acidic and basic pHs [24,28,31], and most of them have not been applied to insulin determination in real biological samples such as skin sweat, which was tested in this work. Moreover, the proposed electrochemical sensor possesses a short response time, low cost and could be used repeatedly at biological pH for more than a month without any significant divergence in its response. 3.6. Analytical applications
found to gradually decrease to 96% and 89% of the initial current, respectively. Such a small decrease of current sensitivity of about 4–11% can be attributed to the high stability of the modified electrode designed.
In order to check its practical application, the proposed sensor was used to determine insulin in its commercial injection (Table 2) and in a skin sweat sample (Table 3) using the recommended procedure. In addition, a certain value of standard solution of
Table 1 Analytical parameters of several modified electrodes for determination of insulin. Electrode
Method
pH
LOD (nM)
Sensitivity (nA/lM)
Dynamic linear range (nM)
Refs.
IrO/GCEa RuRDMs/CEb RuOx/CPEc CNTe/GCE Chitosan-CNT/GCE NiNPs/CCEf
Amp FIA FIAd FIA Amp Amp FIA Amp Amp DPV DPV Amp Amp DPV Amp FIA Microfluidic FIA CV DPV
7.4 7 7.4 7.4 7.4 13
20 2 50 14 30 0.04 0.0026 5 0.036 20 5 0.01 0.22 0.001 500 23 <1000 – 30 3.1
35.2 2.3 102 0.875 48 1.35 102 4 103 2.7 105 – 0.107 864 – 83.9 27.57 0.001 0.441 0.072 1.33 2.4 106 164,333 20
50–500 6–400 100–1000 100–1000 100–3000 0.1–700 0.015–0.1 20–260 0.09–1.4 100–1000 20–1000 0.1–15 0.1–31.5 3.4–68 50–500 100–1000 100–10,000 1.9 105-1.8 106 100–800 10–50
[14] [15] [20] [22] [23] [24]
NiO/MWCNT/SPE Si/CPE SiC/GCE ABNCg/CPE CoO/GCE CNT/Ni/CoO/CNT CNT RuO/RuCN/CFMEh RuOx/CFME MWCNT/DHPi Ru-inorganic polymer film/GCE SiO2NPs-Nafion/GCE a b c d e f g h i
10 2 7.4 10 9 7.5 7.4 7.4 7.4 7.4 2 7.35
Glassy carbon electrode. Ruthenium metallodendrimer multilayers/carbon electrode. Carbon paste electrode. Flow injection analysis. Carbon nanotubes. Carbon ceramic electrode. Acetylene black nanocarbon. Carbon fiber microelectrode. Multi-wall carbon nanotube/Dihydropyran.
[27] [28] [29] [30] [31] [32] [33] [39] [40] [41] [42] This work
N. Amini et al. / Journal of Electroanalytical Chemistry 714-715 (2014) 70–75 Table 3 Determination of insulin in a skin sweat sample. Insulin added (lM) 0.0 0.4 0.6 a b
Insulin found (lM)a 0.218 ± 0.013 0.383 ± 0.036 0.616 ± 0.032
b
Recovery (%) 109.5 95.0 102.0
Average of four determinations ± relative standard deviation. The expected concentration of insulin claimed by the producer was 0.2 lM.
insulin was added into the corresponding samples for testing the recovery. The precision and excellent recoveries reported in Tables 2 and 3 clearly indicated the reliability of the proposed SiO2NPs-Nafion/GCE electrochemical sensor for the insulin determination in real samples of insulin. 4. Conclusion In conclusion, we reported a novel electrochemical sensing strategy for sensitive detection of insulin. The electrooxidation of nanomolar insulin solution was successfully preformed using the GCE modified with SiO2 nanoparticles. The modified electrode shows excellent electrocatalytic activity, antifouling, significantly lower detection limit, grater analytical sensitivity, simple preparation and stability response. The response characteristics of the proposed electrochemical sensor are significantly improved compared to most of some modified electrodes reported as insulin sensors. The results suggested that it is feasible to apply the proposed method to quantitative determination of insulin at physiological pH. Acknowledgments This research was supported by the Iranian Nanotechnology Initiative and Razi University Research Council. References [1] A. Salimi, S. Pourbeyram, H. Hadadzadeh, J. Electroanal. Chem. 542 (2003) 39– 49. [2] A. Arvinte, A.C. Westermann, A. Mariesesay, V. Virtanen, Sens. Actuators, B 150 (2010) 756–763. [3] S.A. Ross, E.A. Gulve, M. Wang, Chem. Rev. 104 (2004) 1255–1282. [4] B.J. Parakasam, S.K. Vareed, L.K. Olson, M.G. Nair, J. Agric. Food. Chem. 53 (2005) 28–31. [5] National Diabetes Fact Sheets: General and National Estimates on Diabetes in the United States, Centers for Disease Control and prevention, 2000. [6] D. Keller, R. Clausen, K. Josefen, J.J. Led, Biochemistry 40 (2001) 10732–10740. [7] C.P. Reis, A.J. Ribeiro, S. Houng, F. Veiga, J. Neufeld, Eur. J. Pharmaceut. Sci. 30 (2007) 392–397. [8] R. Sreekanth, V. Pattabhi, S.S. Rajan, Biochimica 90 (2008) 467–473.
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