by aryl radicals generated from electrochemical reduction of diazonium salts ... reactions. The usefulness of the approach was demonstrated by application to ...
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ScienceDirect Procedia Technology 27 (2017) 187 – 189
Biosensors 2016
Multiplexed electrochemical immunosensor for obesity-related hormones using grafted graphene-modified electrodes as platforms for antibodies immobilization G. Martínez-García, L. Agüí, P. Yáñez-Sedeño*, J.M. Pingarrón Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain
Abstract An electrochemical immunosensor for the simultaneous determination of ghrelin (GHRL) and peptide YY (PYY) using dual screen-printed carbon electrodes modified with reduced graphene oxide (rGO) is presented. Diazonium salt of 4-aminobenzoic acid (4-ABA) was electrochemically grafted on the modified electrodes allowing covalent immobilization of antibodies. After competitive immunoassays using alkaline phosphatase labelled antigens, the affinity reactions were monitored by DPV upon addition of 1-naphthyl phosphate. Calibration plots showed linear current vs. log [hormone] ranges from 10-3 to 100 ng/mL GHRL, and 10-4 to10 ng/mL PYY. The usefulness of dual immunosensor was demonstrated by analysis of spiked human serum and saliva. 2016The TheAuthors. Authors.Published Published Elsevier © 2017 byby Elsevier Ltd.Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016. Peer-review under responsibility of the organizing committee of Biosensors 2016
Keywords: Ghrelin, Peptide YY, obesity, electrochemical immunosensor, multiplex.
1. Introduction Among the various hormones involved in the complex regulation of hunger and saciety, ghrelin (GHRL) and peptide YY (PYY) are destacable since they play important roles at levels of hypothalamus and peripheral circulation exerting orexigen or anorexigen effects. Despite their importance, methods for determining these hormones are scarce. Furthermore, electrochemical grafting consisting of covalent modification of carbon surfaces by aryl radicals generated from electrochemical reduction of diazonium salts has demonstrated to be an excellent strategy for biomolecules immobilization. Here, we report the preparation of a multiplexed electrochemical immunosensor for the determination of obesity-related hormones which satisfies the requirements of sensitivity, selectivity and reproducibility needed for clinical applications. Multiple screen-printed electrodes modified with
2212-0173 © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016 doi:10.1016/j.protcy.2017.04.080
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G. Martínez-García et al. / Procedia Technology 27 (2017) 187 – 189
reduced graphene oxide (rGO) were used as platforms for the immobilization of antibodies. The diazonium salt of 4aminobenzoic acid (4-ABA) was electrochemically grafted at the electrode surfaces, resulting in the covalent attachment of 4-carboxyphenyl to the rGO/GCE. Then, the immunoreagents were covalently immobilized onto the modified electrodes, and competitive immunoassays between the hormones and biotinylated antigens were performed. Differential pulse voltammetry upon 1-naphtylphosphate addition was employed to monitor the affinity reactions. The usefulness of the approach was demonstrated by application to spiked human serum and saliva. 2. Experimental 2.1. Preparation of the immunosensors Reduced graphene oxide (rGO) was firstly obtained. 2 mL of a 1 mg/mL GO aqueous dispersion was sonicated for 120 min, and then centrifuged at 10,000 g for 10 min. After discarding the precipitate, a small volume of 25% NH 3 solution was added to the supernatant solution up to pH 9 -10, and reduction of GO was performed with 2 mM ascorbic acid, by keeping at 95 ºC during 15 min, and rGO dispersion was left in the dark at room temperature. Figure 1 shows the different steps involved in the preparation of the immunosensors. Diazonium salt was prepared by slowly dropping 2 mM NaNO 2 aqueous solution to a 1 mg/mL 4-ABA solution prepared in 1 M HCl and cooled with ice (38 mL NaNO 2 for each 200 mL 4-ABA) (step 1), then alowing to react under stirring during 10 min. Separately, 3 ȝL of rGO were deposited onto each working electrode and, after drying at room temperature, rGOmodified dual electrode was immersed into the diazonium salt solution and ten successive voltammetric cycles from 0 to -1.0 V were scanned. Next, modified SPCEs were washed with water and methanol, and dried at room temperature. For the preparation of the dual GHRL and PYY immunosensor (steps 2 to 4), surface confined carboxylic groups were activated by dropping 3 ȝL of an EDC/NHSS (0.1 M each) aqueous solution onto HOOCPhe-rGO/SPCE 1 and HOOC-Phe-rGO/SPCE 2 , and left to react for 1 h in the dark. After rinsing with water and methanol, the electrodes allowed drying, and 3 ȝ/RIDȝJP/DQWL-GHRL solution and 3 ȝL oIDȝJP/DQWLPYY solution were casted onto the respective electrodes, left standing for 1 h at 37 ºC. Then, 5 ȝL of a 0.2% casein blocking solution were deposited onto each modified electrode and incubated for 1 h at 37 ºC. Competitive immunoassays were carried out (step 3) by dropping 3 ȝL of a mixture solution of GHRL (or the sample) and 0.5 ȝg/mL biotinylated-GHRL (Biotin-GHRL), or 3 ȝL of a mixture of PYY (or the sample) and 0.1 ȝg/mL Biotin-PYY onto anti-GHRL-Phe-rGO/SPCE 1 and anti-PYY-Phr-rGO/SPCE 2 , respectively. After 30 min of incubation at 37 ºC, 10 ȝL of a 5 ȝg/mL AP-Strept were added onto both Biotin-GHRL-anti-GHRL-Phe-rGO/SPCE 1 and Biotin-PYYanti-PYY-Phe-rGO/SPCE 2 , and incubation was performed for 30 min at 37 ºC. Finally (step 4), 45 ȝL of 50 mM Trizma buffer solution, and 5 ȝL of 0.05 M 1-NPP solution were deposited on the dual immunosensor and, after 5 min, differential pulse voltammograms were recorded over the -0.15 to + 0.70 V range. 2.2.. Simultaneous determination of GHRL and PYY in spiked human serum and saliva Lyophilized human serum (S-7394, Sigma) spiked with the hormones at different concentrations was analyzed. After the sample was reconstituted in 1 mL of 0.1 M phosphate buffer solution of pH 7.4, and spiked with the target analytes, the above described procedure was applied and the DPV peak currents were interpolated into the linear portion of the standards calibration graphs. Saliva samples from a lab researcher were collected using a SalivetteR collection device (Sarstedt). The volunteer inserted the cotton swab into the mouth and chewed for 1 min. Then, the swap saturated with saliva was inserted into the vial, sealed with the cap and centrifuged for 5 min at 5000 x g. The determination was performed immediately by diluting 1 mL of spiked saliva with 1 mL 0.1 M Tris buffer of pH 7.2 and applying the procedure described above using 3-mL aliquots of the diluted saliva.
G. Martínez-García et al. / Procedia Technology 27 (2017) 187 – 189
Figure 1. Schematic display of the different steps involved in the preparation and funcioning of the dual GHRL and PYY immunosensors. Reproduced with permission from Elsevier (Ref 1).
3. Results and Discussion Once optimized the experimental variables involved in the preparation of the immunosensors, calibration plots fitted by non-linear regression were obtained by DPV for both hormones in the 10-7 to 104 ng/mL concentration range. Least square fitting of the curves provided also linear current vs. log [hormone] plots extending between 10-3 and 100 ng/mL GHRL, and 10-4 and 10 ng/mL PYY. These ranges are adequate for the determination of both hormones at clinical levels in serum and saliva. The limits of detection, calculated as the lowest concentrations that could be differentiated from zero, were 1.0 pg/mL GHRL and 0.02 pg/mL PYY. Reproducibility was also evaluated by measuring 0.1 ng/mL GHRL or 0.01 ng/mL PYY with different dual immunosensors prepared on the same day (n=10), or in different days (n=8). The RSD values obtained were 2.9% and 2.8% (GHRL), and 2.4% and 2.9% (PYY). The storage stability of the anti-GHRL-Phe-rGO/SPCE and anti- PYY-Phe-rGO/SPCE conjugates was also investigated. Various dual bioelectrodes were prepared on the same day and stored in a refrigerator at 4 ºC. Each immunosensor was used to measure the voltammetric response for 0.1 ng/mL GHRL or 0.01 ng/mL PYY. A high storage stability of the prepared configurations, with the DPV peak currents remaining within the control limits set at ±3 x standard deviation of the responses obtained on the first day of the study, for at least 10 days (no longer period of storage time was tested). The developed dual immunosensor also present an excellent selectivity for both hormones against other proteins (adiponectin, insulin, human growth hormone, deacylated GHRL, or follicle stimulant hormone (FSH). Moreover, cross-talking between the two target hormones did not occur. As no significative differences between the slope values in the samples solutions and the standards were observed, the determination of GHRL and PYY in spiked serum or saliva was accomplished by simple interpolation of the measured ip values into the respective standards calibration plot. The results obtained for different target analyte concentration levels: 175, 65 or 6.5 pg/mL GHRL, and 3.5, 17.5 or 35 pg/mL PYY in serum, or 95 pg/mL GHRL and 37.5 pg/mL PYY in saliva, provided recovery values ranging between 96 and 103%, with no significant differences due to the different nature of biological sample analyzed, thus demonstrating the suitability of the approach for the accurate analysis of these two biomarkers in human serum and saliva. References [1] G. Martínez-García, L. Agüí, P. Yáñez-Sedeño, J.M. Pingarrón, Electrochim. Acta, 202 (2016) 209
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