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Adsorptive stripping voltammetry of indigo blue in a flow system
C H A P T E R
5 Adsorptive stripping voltammetry of indigo blue in a flow system Estefanía Costa-Rama1, 2, M. Teresa Fernández Abedul2 1
REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal; 2Departamento de Química Física y Analítica, Universidad de Oviedo, Oviedo, Spain
5.1 Background Adsorptive stripping voltammetry (AdSV) is a variation of stripping voltammetry which refers to the stripping of species spontaneously adsorbed on the surface of the working electrode without needing a previous electrolysis step [1]. In Chapter 4, anodic stripping voltammetry is presented for determination of lead and cadmium. A cathodic electrodeposition of metals allows further quantification through their anodic stripping. In this case, adsorption is the mechanism for the preconcentration on the electrode surface. For the stripping step, different voltammetric techniques, such as linear sweep, differential pulse, or square wave voltammetry, can be used. In this experiment, alternating current voltammetry (ACV) is used as detection technique for the stripping step. Hence, the potential program imposed on the working electrode is a direct current (dc) mean value, Edc, which is scanned slowly with time, plus a sinusoidal component, Eac. The alternating current (ac) signal thus causes a perturbation in the surface concentration, around the concentration maintained by the dc potential ramp [2]. The measured responses are the magnitude of the ac component of the current at the frequency of Eac and its phase angle with respect to Eac. The ac voltammogram shows a peak, which height is proportional to the concentration of the analyte and, for a reversible reaction, to the square root of the frequency [2]: ip ¼
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00005-X
n2 F2 Au1=2 D1=2 C DE 4RT
47
Copyright © 2020 Elsevier Inc. All rights reserved.
48
5. Adsorptive stripping voltammetry of indigo blue in a flow system
Eac Eac or iac
iac 2p/w (2p+f )/w
0
p/w
t
FIGURE 5.1 Relationship between alternating current and voltage signals at frequency u. Reprinted with modifications from A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, second ed., John Wiley & Sons: New York, 2001.
where the term DE is the amplitude and u is the frequency. In Fig. 5.1 the relationship between the alternating current and voltage signals at frequency u is shown. The detection of the ac component allows separating between the faradaic and capacitive components of the current because the phase shift of the faradaic component, relative to the applied sinusoidal potential, is different from this of the capacitive. Then, it could be decreased if a phase-sensitive detection is employed. In this experiment, the detection is integrated in a flow injection analysis (FIA) system to inject the product of an enzyme-linked immunosorbent assay (ELISA) for interleukin-10 (IL-10) quantification. FIA, also commented in Chapters 9 and 28, consists of the injection of a fixed volume of sample into a nonsegmented continuous laminar flow of a carrier solution that transports the sample to the detector [3,4]. Thus, the FIA system allows simple and flexible configuration that helps to the automatization of analysis, decreasing analysis time and human errors. The configuration in this case is basic, as no reaction occurs inside the system. Then, peristaltic pump, injector (rotary valve), tubing, and detector are the components of the FIA system. The two main configurations for the flow cell are wall jet and thin layer. In the first one, the sample enters the cell perpendicularly to the working electrode, meanwhile in the thin-layer model, the flow passes over the electrode, entering laterally from one side and leaving by the opposite. ELISAs are among the most common immunoassays that are performed every day. The use of microplates allows parallelization of assays, and the employ of enzymes as labels (there are also many other possible [5e7], e.g., metallic nanoparticles or organic redox compounds, see Chapter 20) has resulted in very sensitive methodologies. In this case, among the several immunoassay formats that exist, a sandwich type has been chosen. It is based on the interaction of the antigen with a specific antibody immobilized onto the surface of the wells. Then, an enzyme-labeled antibody is added and it binds with the antigen forming a “sandwich.” A substrate is later enzymatically converted into an electroactive product. Alkaline phosphatase is an enzyme of particular interest because of its broad substrate specificity and high turnover number. Several substrates have been employed: phenyl, p-nitrophenyl, p-aminophenyl,
I. Dynamic electroanalytical techniques
49
5.1 Background
naphthyl phosphate, etc. The product of the ideal substrate has to give rise to a sensitive oxidation at low potentials, which enhances selectivity, together with the easy elimination from the electrode of the products generated, which increases precision. 3-Indoxyl phosphate (3-IP) fulfills all these requirements and has been proposed as an appropriate alkaline phosphatase substrate for enzyme immunoassays with voltammetric detection [8]. The adsorption behavior of indigo allows the use of an adsorptive stripping voltammetric methodology, which enhances sensitivity and selectivity. The enzymatic reaction (Fig. 5.2) comprises the hydrolysis of the phosphate moiety by alkaline phosphatase (AP), the formation of the unstable enol product and its subsequent oxidation in air to give indigo blue. Indigo blue is nonsoluble in aqueous solutions and it can be solubilized adding sulfuric acid that converts it into indigo carmine. In this experiment, adapted from Ref. [10], an ELISA is performed for detection of IL-10. This is an antiinflammatory cytokine that plays a relevant role in the infection process by regulation of the immune response [11]. Elevated levels of IL-10 indicate an acute stage of inflammation and are also found in patients of malignant tumors such as melanoma, colorectal, ovarian, breast, or gastric carcinomas [12]. The enzyme AP is used as label in the ELISA assay and it catalyzes the hydrolysis of 3-IP to indigo blue. This enzymatic product, once converted into indigo carmine after stopping the reaction with sulfuric acid, is introduced into the FIA system that possesses an electrochemical cell with a carbon paste working electrode. Indigo is adsorbed on the surface of the working electrode and, then, it is quantified by stripping voltammetry.
H
H
N
N
+ H2O O-
O- O
AP
+ HPO42-
Basic medium 9 < pH < 10
O-
P Ceto-enol equilibrium
O 3-indoxyl phosphate H N
Oxidation
H2O2 + O
H
O
N H
Indigo blue
FIGURE 5.2
N
O Indoxyl group
Alkaline phosphatase (AP) hydrolysis of 3-indoxyl phosphate to produce indigo blue.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
5.2 Chemicals and supplies Enzyme-linked immunosorbent assay e 0.05 M carbonate buffer pH 9.6 containing 0.1% NaN3 (coating solution). e 0.1 M Tris-HCl buffer with 150 mM NaCl (TBS) and 0.05% Tween 20 (TW), 0.05% NaN3, and 2% casein pH 7.4 (blocking solution). e 0.1 M TBS 7.4 containing 0.02% NaN3 and 0.05% TW (washing buffer). e Monoclonal anti-IL-10 antibody (in 0.1 M phosphate buffer, 150 mM NaCl pH 7.4). e Il-10 (dilutions prepared in 0.1 M TBS, 0.05% NaN3, 0.05% TW, 1% casein, pH 7.4). e Biotinylated anti-IL-10 antibody (in 20 mM TBS, 0.1% BSA pH 7.4). e AP labeled with streptavidin (prepared in 10 mM Tris-HCl buffer, 50 mM NaCl, 1.5 mM MgCl2, 0.2% TW pH 7.4). e 3-IP (dilutions prepared in 0.1 M Tris-HCl buffer pH 9.8 with 10 mM MgCl2, stored at 4 C protected from light). e H2SO4 (concentrated and 1 M solutions). e Microtiter plates. e Stirrer with microtiter adapter. e Incubator. Detection e e e e e e e e e
Peristaltic pump with two channels Six-port rotary valve with 20- and 100-mL loops. PVC tubes. Thin-layer flow cell. Graphite powder. Paraffin oil. Ag/AgCl/saturated KCl as reference electrode. Stainless steel tube as counter electrode. Potentiostat and computer system. General chemicals and materials
e Indigo carmine (diluted in 0.1 M H2SO4) e 100-mL eight-channel micropipette and corresponding tips e 1.5-mL microcentrifuge tubes, pH-meter, and analytical balance Ultrapure water is employed throughout the work to prepare solutions and to clean electrodes and glassware.
5.3 Hazards Sodium azide is very toxic if swallowed or in contact with skin. Read its safety data sheet and handle it in a fume hood. Students are required to wear a lab coat, appropriate gloves and eye protection, as well as to handle all the reagents with care, especially concentrated acids and bases as well as sodium azide.
I. Dynamic electroanalytical techniques
5.5 Experimental procedures
51
5.4 Flow injection analysis electrochemical system First, the FIA system (Fig. 5.3) is prepared connecting the pump, the valve, and the flow cell by the PVC tubing. The sample in introduced in the FIA system by aspiration from the well of the microtiter plate. More information about FIA systems can be found in Chapters 9 and 28. The electrochemical thin-layer flow cell was equipped with a carbon paste electrode (geometric area of 9.6 mm2) and consists of two methacrylate blocks and a PVC spacer put together with four screws. The carbon paste was prepared by intimately mixing graphite powder (1 g) and paraffin oil (0.36 mL). This carbon paste is packed in the corresponding place of the flow cell and the surface can be entirely or partially renewed with the help of a piston inserted in the electrode body. The reference and counter electrodes are located downstream in a syringe coupled to the cell outlet. The counter electrode is a hollow stainless steel tube that leads to the waste.
5.5 Experimental procedures 5.5.1 Enzyme-linked immunosorbent assay procedure To perform the ELISA assay, represented in Fig. 5.3A, several steps have to be followed: 1. Coat each well with 100 mL of anti-IL-10 antibody (6 mg/mL) and incubate overnight at room temperature. Wash the plate with washing solution four times using 200 mL per well. 2. Add 200 mL of the blocking buffer and incubate at 37 C for 3 h and rinse again carefully.
FIGURE 5.3 Schematic representation of (A) the enzyme-linked immunosorbent assay (ELISA) and (B) the flow injection analysis (FIA) system. 3-IP, 3-indoxyl phosphate; AP, alkaline phosphatase.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
3. Deposit 100 mL of IL-10 (analyte) to each well and incubate overnight at room temperature and then, wash. 4. Add 100 mL of biotinylated anti-IL-10 antibody (1 mg/mL), keep at room temperature for 2 h, and perform the washing step. 5. Incubate with streptavidin-AP (1:1000 diluted) for 1 h at room temperature and rinse again. 6. Add 100 mL of 0.2 mM 3-IP solution to each well. The enzymatic reaction occurs for 2 h at 35 C and then it is stopped by adding 150 mL of H2SO4 concentrated. 7. Wait for 10 min and then aspire the content of the well, which contains the generated indigo, using a capillary connected to the tubing of the peristaltic pump until the loop of the injection valve is filled. 8. Inject the sample into the FIA system (“load” position) to carry out the voltammetric detection.
5.5.2 Flow procedure and voltammetric detection 1. Pump a stream of 1 M H2SO4 (carrier) at 2 mL/min through the FIA system and confirm the absence of bubbles. 2. Pretreat the surface of the working electrode by applying þ1.3 V for 30 s. 3. After having filled the loop, change the injection valve from “load” to “inject” position to introduce indigo into the flow and change the potential to 0.3 V. In this step, indigo is adsorbed on the surface of the working electrode. 4. Pass the carrier through the cell for a fixed time (e.g, 15 s). 5. Stop the flow and scan the potential in the positive direction to þ0.9 V in the ACV format (e.g., 75 Hz of frequency, 35 mV of amplitude, and 0 degree phase angle) to obtain the corresponding voltammogram.
5.5.3 Electrochemical behavior of indigo As indigo is the product of the enzymatic reaction that is measured in the FIA system, it is interesting to know its electrochemical behavior (see additional note 2 in Section 5.7). To know which is the analytical signal, inject 100 mL of a 5-mM indigo carmine solution in the FIA system (using the injection valve) and record the corresponding ACV (record first an ACV after injecting the carrier to obtain the background). Two well-defined peaks must be obtained (Fig. 5.4). Choose one of the peaks for the quantification and justify it.
5.5.4 Calibration curve of IL-10 Obtain a calibration curve for IL-10 in the concentration range from 0.2 to 1500 pg/mL. Represent the intensity of the peak current vs. the concentration (Fig. 5.5) and also the loge log graph. Calculate the linear range, the sensitivity, the limit of detection, and the limit of quantification.
I. Dynamic electroanalytical techniques
5.6 Lab report
53
FIGURE 5.4 Example of the alternating current voltammogram obtained for indigo carmine. Reprinted with modifications from M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62.
5.6 Lab report At the end of the experiment, write a lab report including introduction, experimental part (reagents, materials, equipment, and protocols), results and discussion, and conclusions. The following points should be considered to write the report: 1. In the introduction, explain the purpose and the basis of the experiment. Include an overview on the different types of immunoassays. Explain the advantages and disadvantages of the use of labels in immunosensors (vs. label-free approaches) and how the label here used allows obtaining the analytical signal. 2. Detail the protocol followed including schemes. 3. Discuss the results obtained and include graphs and tables with representative raw data. 4. For the calibration plot, indicate the values obtained for the figures of merit. 5. Discuss the incidences that happened during the experiment and the main conclusions.
ip (µA)
[IL-10] (pg/mL) FIGURE 5.5
Example of a calibration plot obtained for interleukin-10 (IL-10). Reprinted with modifications from M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
5.7 Additional notes 1. 3-IP solutions must be prepared the day of use and kept away form light. 2. The electrochemical behavior of indigo carmine (product of the enzymatic reaction after stopping with sulfuric acid) could be studied employing a conventional cell of three electrodes and recording the corresponding cyclic voltammograms (CVs) [13]. Alternatively, screen-printed electrodes could be also employed. The comparison between electrodes is interesting (e.g., diffusional behavior on screen-printed electrodes and adsorptive behavior on carbon paste electrodes). In Fig. 5.6, an example of the CV recorded in 0.1 M HClO4 on screen-printed electrodes is presented. 3. Screen-printed electrodes can be used also in a flow cell [14]. As indigo presents a diffusional behavior on these electrodes and the flow cleans the electrode surface, even being disposable, they can be reused. 4. ACVs of indigo carmine show two peaks at around 0.1 and þ0.7 V. The first one is due to the oxidation of leucoindigo to indigo and the second one to the conversion of indigo to dehydroindigo. The first one is chosen for quantification because it shows a higher peak current and appears at lower potential. A voltammogram has to be recorded always in the background for comparison. 5. The different parameters of ACV should be optimized (especially the phase angle). 6. The calibration curve (ip vs. [IL-10]) is fitted to a log plot to obtain a linear equation in the whole range of concentrations (from 0.2 to 1500 pg/mL). Different functions can be evaluated. 7. A commercial protein matrix could be used to validate the methodology, spiking it with different concentrations of IL-10. 8. A spectrophotometric detection could be used to compare the results obtained by the electrochemical detection using p-nitrophenylphosphate (pNPP), instead of 3-IP, as enzyme substrate. When pNPP reacts with the enzyme AP, a yellow color is developed allowing the calibration by measuring the absorbance at 405 nm. To carry out the spectrophotometric detection, 100 mL of 1-mM pNPP solution (prepared in 0.1 M Tris-HCl buffer pH 9.8 containing 10 mM of MgCl2) is added to each well. The
FIGURE 5.6 Cyclic voltammetric responses recorded on (A) 0.1 M HClO4 and (B) indigo carmine solution in 0.1 M HClO4. Reprinted with modifications from M.J. Bengoechea Álvarez, M.T. Fernández Abedul, A. Costa García, Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes, Anal. Chim. Acta 462 (2002) 31e37.
I. Dynamic electroanalytical techniques
References
9. 10.
11. 12.
55
enzymatic reaction occurs for 30 min at 35 C and then it is stopped by adding 150 mL of 1 M NaOH into each well. Finally, absorbances are read at 405 nm. Immunoassays (ELISA or other format) can be performed for other analytes (e.g., pneumolysin [8,9]) using AP-labeled antibodies. In this experiment, an immunoassay with introduction of the product in a flow system is employed. The flow system acts as a linkage between the batch immunoassay and the detection system. The coupling of the flow system implies an important advantage when compared with batch protocols of immunoassays that use AP as label and detect the enzymatic product of 3-IP: (i) The electrode pretreatment commonly necessary for obtaining a reproducible methodology is simpler because the flow acts cleaning its surface; (ii) on the other hand, the accumulation step is reduced to the passage of the substrate loop by the electrode so that analysis time is decreased, and (iii) as the measurement is performed after the adsorption in fresh electrolyte, the influence of interfering species can be eliminated. However, AdSV and ACV could be demonstrated in a simpler way without requiring an immunoassay and a flow system as exemplified for other analytes such as melatonin [15]. This analyte (considered in Chapter 9) presents a reversible pair, whose current is increased after accumulation. It seems that the system can be a very interesting alternative and very appropriate also to study adsorption processes. The experiment combines immunoassay/flow/adsorptive stripping/ACV. Discussion of possible combinations with only two or three elements can be very interesting. 3-IP could be employed also as a substrate for other enzyme, also employed commonly in enzymatic immunoassays, horseradish peroxidase [16].
5.8 Assessment and discussion questions 1. Explain the basis of the electrochemical technique employed for the detection and suggest other possibilities. 2. What is the format of the immunoassay employed and what are the main steps? 3. Draw a scheme for the FIA system. 4. What is the difference between indigo blue and indigo carmine? 5. Explain the electrochemical behavior of indigo carmine. 6. Compare the analytical characteristics of this method with others found in the bibliography for the same or similar analytes, using other labels and methodologies. 7. Look for other electrochemical immunosensors that use alkaline phosphatase as label and discuss their differences.
References [1] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, second ed., John Wiley & Sons, New York, 2001. [2] J. Wang, Analytical Electrochemistry, second ed., Wiley-VCH, New York, 2000. [3] A.A. Kulkarni, I.S. Vaidya, Flow injection analysis: an overview, J. Crit. Rev. 2 (2015) 19e24. [4] M. Trojanowicz, K. Kołaci nska, Recent advances in flow injection analysis, Analyst 141 (2016) 2085e2139.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
[5] C. Kokkinos, A. Economou, M.I. Prodromidis, Electrochemical immunosensors: critical survey of different architectures and transduction strategies, TrAC Trends Anal. Chem. 79 (2016) 88e105. [6] F.S. Felix, L. Angnes, Electrochemical immunosensors e a powerful tool for analytical applications, Biosens. Bioelectron. 102 (2018) 470e478. [7] E.C. Rama, A. Costa-García, Screen-printed electrochemical immunosensors for the detection of cancer and cardiovascular biomarkers, Electroanalysis 28 (2016). [8] C. Fernández-Sánchez, M.B. González-García, A. Costa-García, 3-Indoxyl phosphate: an alkaline phosphatase substrate for enzyme immunoassays with voltammetric detection, Electroanalysis 10 (1998) 249e255. [9] C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Pneumolysin ELISA with adsorptive voltammetric detection of indigo in a flow system, Electroanalysis 13 (2001) 559e566. [10] M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62. [11] M. Saraiva, A.O. Garra, The regulation of IL-10 production by immune cells, Nat. Rev. Immunol. 10 (2010) 170e181. [12] A.O. Garra, F.J. Barrat, A.G. Castro, A. Vicari, C. Hawrylowicz, Strategies for use of IL-10 or its antagonists in human disease, Immunol. Rev. 223 (2008) 114e131. [13] C. Fernández-Sánchez, A. Costa-García, Voltammetric studies of indigo adsorbed on pre-treated carbon paste electrodes, Electrochem. Comm. 2 (2000) 776e781. [14] M.J. Bengoechea Álvarez, M.T. Fernández Abedul, A. Costa García, Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes, Anal. Chim. Acta 462 (2002) 31e37. [15] J.L. Corujo-Antuña, S. Martínez-Montequín, M.T. Fernández-Abedul, A. Costa-García, Sensitive adsorptive stripping voltammetric methodologies for the determination of melatonin in biological fluids, Electroanalysis 15 (2003) 773e778. [16] P. Fanjul-Bolado, M.B. González-García, A. Costa-García, 3-Indoxyl phosphate as an electrochemical substrate for horseradish peroxidase, Electroanalysis 16 (2004) 988e993.
I. Dynamic electroanalytical techniques
5 Adsorptive stripping voltammetry of indigo blue in a flow system Estefanía Costa-Rama1, 2, M. Teresa Fernández Abedul2 1
REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal; 2Departamento de Química Física y Analítica, Universidad de Oviedo, Oviedo, Spain
5.1 Background Adsorptive stripping voltammetry (AdSV) is a variation of stripping voltammetry which refers to the stripping of species spontaneously adsorbed on the surface of the working electrode without needing a previous electrolysis step [1]. In Chapter 4, anodic stripping voltammetry is presented for determination of lead and cadmium. A cathodic electrodeposition of metals allows further quantification through their anodic stripping. In this case, adsorption is the mechanism for the preconcentration on the electrode surface. For the stripping step, different voltammetric techniques, such as linear sweep, differential pulse, or square wave voltammetry, can be used. In this experiment, alternating current voltammetry (ACV) is used as detection technique for the stripping step. Hence, the potential program imposed on the working electrode is a direct current (dc) mean value, Edc, which is scanned slowly with time, plus a sinusoidal component, Eac. The alternating current (ac) signal thus causes a perturbation in the surface concentration, around the concentration maintained by the dc potential ramp [2]. The measured responses are the magnitude of the ac component of the current at the frequency of Eac and its phase angle with respect to Eac. The ac voltammogram shows a peak, which height is proportional to the concentration of the analyte and, for a reversible reaction, to the square root of the frequency [2]: ip ¼
Laboratory Methods in Dynamic Electroanalysis https://doi.org/10.1016/B978-0-12-815932-3.00005-X
n2 F2 Au1=2 D1=2 C DE 4RT
47
Copyright © 2020 Elsevier Inc. All rights reserved.
48
5. Adsorptive stripping voltammetry of indigo blue in a flow system
Eac Eac or iac
iac 2p/w (2p+f )/w
0
p/w
t
FIGURE 5.1 Relationship between alternating current and voltage signals at frequency u. Reprinted with modifications from A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, second ed., John Wiley & Sons: New York, 2001.
where the term DE is the amplitude and u is the frequency. In Fig. 5.1 the relationship between the alternating current and voltage signals at frequency u is shown. The detection of the ac component allows separating between the faradaic and capacitive components of the current because the phase shift of the faradaic component, relative to the applied sinusoidal potential, is different from this of the capacitive. Then, it could be decreased if a phase-sensitive detection is employed. In this experiment, the detection is integrated in a flow injection analysis (FIA) system to inject the product of an enzyme-linked immunosorbent assay (ELISA) for interleukin-10 (IL-10) quantification. FIA, also commented in Chapters 9 and 28, consists of the injection of a fixed volume of sample into a nonsegmented continuous laminar flow of a carrier solution that transports the sample to the detector [3,4]. Thus, the FIA system allows simple and flexible configuration that helps to the automatization of analysis, decreasing analysis time and human errors. The configuration in this case is basic, as no reaction occurs inside the system. Then, peristaltic pump, injector (rotary valve), tubing, and detector are the components of the FIA system. The two main configurations for the flow cell are wall jet and thin layer. In the first one, the sample enters the cell perpendicularly to the working electrode, meanwhile in the thin-layer model, the flow passes over the electrode, entering laterally from one side and leaving by the opposite. ELISAs are among the most common immunoassays that are performed every day. The use of microplates allows parallelization of assays, and the employ of enzymes as labels (there are also many other possible [5e7], e.g., metallic nanoparticles or organic redox compounds, see Chapter 20) has resulted in very sensitive methodologies. In this case, among the several immunoassay formats that exist, a sandwich type has been chosen. It is based on the interaction of the antigen with a specific antibody immobilized onto the surface of the wells. Then, an enzyme-labeled antibody is added and it binds with the antigen forming a “sandwich.” A substrate is later enzymatically converted into an electroactive product. Alkaline phosphatase is an enzyme of particular interest because of its broad substrate specificity and high turnover number. Several substrates have been employed: phenyl, p-nitrophenyl, p-aminophenyl,
I. Dynamic electroanalytical techniques
49
5.1 Background
naphthyl phosphate, etc. The product of the ideal substrate has to give rise to a sensitive oxidation at low potentials, which enhances selectivity, together with the easy elimination from the electrode of the products generated, which increases precision. 3-Indoxyl phosphate (3-IP) fulfills all these requirements and has been proposed as an appropriate alkaline phosphatase substrate for enzyme immunoassays with voltammetric detection [8]. The adsorption behavior of indigo allows the use of an adsorptive stripping voltammetric methodology, which enhances sensitivity and selectivity. The enzymatic reaction (Fig. 5.2) comprises the hydrolysis of the phosphate moiety by alkaline phosphatase (AP), the formation of the unstable enol product and its subsequent oxidation in air to give indigo blue. Indigo blue is nonsoluble in aqueous solutions and it can be solubilized adding sulfuric acid that converts it into indigo carmine. In this experiment, adapted from Ref. [10], an ELISA is performed for detection of IL-10. This is an antiinflammatory cytokine that plays a relevant role in the infection process by regulation of the immune response [11]. Elevated levels of IL-10 indicate an acute stage of inflammation and are also found in patients of malignant tumors such as melanoma, colorectal, ovarian, breast, or gastric carcinomas [12]. The enzyme AP is used as label in the ELISA assay and it catalyzes the hydrolysis of 3-IP to indigo blue. This enzymatic product, once converted into indigo carmine after stopping the reaction with sulfuric acid, is introduced into the FIA system that possesses an electrochemical cell with a carbon paste working electrode. Indigo is adsorbed on the surface of the working electrode and, then, it is quantified by stripping voltammetry.
H
H
N
N
+ H2O O-
O- O
AP
+ HPO42-
Basic medium 9 < pH < 10
O-
P Ceto-enol equilibrium
O 3-indoxyl phosphate H N
Oxidation
H2O2 + O
H
O
N H
Indigo blue
FIGURE 5.2
N
O Indoxyl group
Alkaline phosphatase (AP) hydrolysis of 3-indoxyl phosphate to produce indigo blue.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
5.2 Chemicals and supplies Enzyme-linked immunosorbent assay e 0.05 M carbonate buffer pH 9.6 containing 0.1% NaN3 (coating solution). e 0.1 M Tris-HCl buffer with 150 mM NaCl (TBS) and 0.05% Tween 20 (TW), 0.05% NaN3, and 2% casein pH 7.4 (blocking solution). e 0.1 M TBS 7.4 containing 0.02% NaN3 and 0.05% TW (washing buffer). e Monoclonal anti-IL-10 antibody (in 0.1 M phosphate buffer, 150 mM NaCl pH 7.4). e Il-10 (dilutions prepared in 0.1 M TBS, 0.05% NaN3, 0.05% TW, 1% casein, pH 7.4). e Biotinylated anti-IL-10 antibody (in 20 mM TBS, 0.1% BSA pH 7.4). e AP labeled with streptavidin (prepared in 10 mM Tris-HCl buffer, 50 mM NaCl, 1.5 mM MgCl2, 0.2% TW pH 7.4). e 3-IP (dilutions prepared in 0.1 M Tris-HCl buffer pH 9.8 with 10 mM MgCl2, stored at 4 C protected from light). e H2SO4 (concentrated and 1 M solutions). e Microtiter plates. e Stirrer with microtiter adapter. e Incubator. Detection e e e e e e e e e
Peristaltic pump with two channels Six-port rotary valve with 20- and 100-mL loops. PVC tubes. Thin-layer flow cell. Graphite powder. Paraffin oil. Ag/AgCl/saturated KCl as reference electrode. Stainless steel tube as counter electrode. Potentiostat and computer system. General chemicals and materials
e Indigo carmine (diluted in 0.1 M H2SO4) e 100-mL eight-channel micropipette and corresponding tips e 1.5-mL microcentrifuge tubes, pH-meter, and analytical balance Ultrapure water is employed throughout the work to prepare solutions and to clean electrodes and glassware.
5.3 Hazards Sodium azide is very toxic if swallowed or in contact with skin. Read its safety data sheet and handle it in a fume hood. Students are required to wear a lab coat, appropriate gloves and eye protection, as well as to handle all the reagents with care, especially concentrated acids and bases as well as sodium azide.
I. Dynamic electroanalytical techniques
5.5 Experimental procedures
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5.4 Flow injection analysis electrochemical system First, the FIA system (Fig. 5.3) is prepared connecting the pump, the valve, and the flow cell by the PVC tubing. The sample in introduced in the FIA system by aspiration from the well of the microtiter plate. More information about FIA systems can be found in Chapters 9 and 28. The electrochemical thin-layer flow cell was equipped with a carbon paste electrode (geometric area of 9.6 mm2) and consists of two methacrylate blocks and a PVC spacer put together with four screws. The carbon paste was prepared by intimately mixing graphite powder (1 g) and paraffin oil (0.36 mL). This carbon paste is packed in the corresponding place of the flow cell and the surface can be entirely or partially renewed with the help of a piston inserted in the electrode body. The reference and counter electrodes are located downstream in a syringe coupled to the cell outlet. The counter electrode is a hollow stainless steel tube that leads to the waste.
5.5 Experimental procedures 5.5.1 Enzyme-linked immunosorbent assay procedure To perform the ELISA assay, represented in Fig. 5.3A, several steps have to be followed: 1. Coat each well with 100 mL of anti-IL-10 antibody (6 mg/mL) and incubate overnight at room temperature. Wash the plate with washing solution four times using 200 mL per well. 2. Add 200 mL of the blocking buffer and incubate at 37 C for 3 h and rinse again carefully.
FIGURE 5.3 Schematic representation of (A) the enzyme-linked immunosorbent assay (ELISA) and (B) the flow injection analysis (FIA) system. 3-IP, 3-indoxyl phosphate; AP, alkaline phosphatase.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
3. Deposit 100 mL of IL-10 (analyte) to each well and incubate overnight at room temperature and then, wash. 4. Add 100 mL of biotinylated anti-IL-10 antibody (1 mg/mL), keep at room temperature for 2 h, and perform the washing step. 5. Incubate with streptavidin-AP (1:1000 diluted) for 1 h at room temperature and rinse again. 6. Add 100 mL of 0.2 mM 3-IP solution to each well. The enzymatic reaction occurs for 2 h at 35 C and then it is stopped by adding 150 mL of H2SO4 concentrated. 7. Wait for 10 min and then aspire the content of the well, which contains the generated indigo, using a capillary connected to the tubing of the peristaltic pump until the loop of the injection valve is filled. 8. Inject the sample into the FIA system (“load” position) to carry out the voltammetric detection.
5.5.2 Flow procedure and voltammetric detection 1. Pump a stream of 1 M H2SO4 (carrier) at 2 mL/min through the FIA system and confirm the absence of bubbles. 2. Pretreat the surface of the working electrode by applying þ1.3 V for 30 s. 3. After having filled the loop, change the injection valve from “load” to “inject” position to introduce indigo into the flow and change the potential to 0.3 V. In this step, indigo is adsorbed on the surface of the working electrode. 4. Pass the carrier through the cell for a fixed time (e.g, 15 s). 5. Stop the flow and scan the potential in the positive direction to þ0.9 V in the ACV format (e.g., 75 Hz of frequency, 35 mV of amplitude, and 0 degree phase angle) to obtain the corresponding voltammogram.
5.5.3 Electrochemical behavior of indigo As indigo is the product of the enzymatic reaction that is measured in the FIA system, it is interesting to know its electrochemical behavior (see additional note 2 in Section 5.7). To know which is the analytical signal, inject 100 mL of a 5-mM indigo carmine solution in the FIA system (using the injection valve) and record the corresponding ACV (record first an ACV after injecting the carrier to obtain the background). Two well-defined peaks must be obtained (Fig. 5.4). Choose one of the peaks for the quantification and justify it.
5.5.4 Calibration curve of IL-10 Obtain a calibration curve for IL-10 in the concentration range from 0.2 to 1500 pg/mL. Represent the intensity of the peak current vs. the concentration (Fig. 5.5) and also the loge log graph. Calculate the linear range, the sensitivity, the limit of detection, and the limit of quantification.
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5.6 Lab report
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FIGURE 5.4 Example of the alternating current voltammogram obtained for indigo carmine. Reprinted with modifications from M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62.
5.6 Lab report At the end of the experiment, write a lab report including introduction, experimental part (reagents, materials, equipment, and protocols), results and discussion, and conclusions. The following points should be considered to write the report: 1. In the introduction, explain the purpose and the basis of the experiment. Include an overview on the different types of immunoassays. Explain the advantages and disadvantages of the use of labels in immunosensors (vs. label-free approaches) and how the label here used allows obtaining the analytical signal. 2. Detail the protocol followed including schemes. 3. Discuss the results obtained and include graphs and tables with representative raw data. 4. For the calibration plot, indicate the values obtained for the figures of merit. 5. Discuss the incidences that happened during the experiment and the main conclusions.
ip (µA)
[IL-10] (pg/mL) FIGURE 5.5
Example of a calibration plot obtained for interleukin-10 (IL-10). Reprinted with modifications from M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62.
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5. Adsorptive stripping voltammetry of indigo blue in a flow system
5.7 Additional notes 1. 3-IP solutions must be prepared the day of use and kept away form light. 2. The electrochemical behavior of indigo carmine (product of the enzymatic reaction after stopping with sulfuric acid) could be studied employing a conventional cell of three electrodes and recording the corresponding cyclic voltammograms (CVs) [13]. Alternatively, screen-printed electrodes could be also employed. The comparison between electrodes is interesting (e.g., diffusional behavior on screen-printed electrodes and adsorptive behavior on carbon paste electrodes). In Fig. 5.6, an example of the CV recorded in 0.1 M HClO4 on screen-printed electrodes is presented. 3. Screen-printed electrodes can be used also in a flow cell [14]. As indigo presents a diffusional behavior on these electrodes and the flow cleans the electrode surface, even being disposable, they can be reused. 4. ACVs of indigo carmine show two peaks at around 0.1 and þ0.7 V. The first one is due to the oxidation of leucoindigo to indigo and the second one to the conversion of indigo to dehydroindigo. The first one is chosen for quantification because it shows a higher peak current and appears at lower potential. A voltammogram has to be recorded always in the background for comparison. 5. The different parameters of ACV should be optimized (especially the phase angle). 6. The calibration curve (ip vs. [IL-10]) is fitted to a log plot to obtain a linear equation in the whole range of concentrations (from 0.2 to 1500 pg/mL). Different functions can be evaluated. 7. A commercial protein matrix could be used to validate the methodology, spiking it with different concentrations of IL-10. 8. A spectrophotometric detection could be used to compare the results obtained by the electrochemical detection using p-nitrophenylphosphate (pNPP), instead of 3-IP, as enzyme substrate. When pNPP reacts with the enzyme AP, a yellow color is developed allowing the calibration by measuring the absorbance at 405 nm. To carry out the spectrophotometric detection, 100 mL of 1-mM pNPP solution (prepared in 0.1 M Tris-HCl buffer pH 9.8 containing 10 mM of MgCl2) is added to each well. The
FIGURE 5.6 Cyclic voltammetric responses recorded on (A) 0.1 M HClO4 and (B) indigo carmine solution in 0.1 M HClO4. Reprinted with modifications from M.J. Bengoechea Álvarez, M.T. Fernández Abedul, A. Costa García, Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes, Anal. Chim. Acta 462 (2002) 31e37.
I. Dynamic electroanalytical techniques
References
9. 10.
11. 12.
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enzymatic reaction occurs for 30 min at 35 C and then it is stopped by adding 150 mL of 1 M NaOH into each well. Finally, absorbances are read at 405 nm. Immunoassays (ELISA or other format) can be performed for other analytes (e.g., pneumolysin [8,9]) using AP-labeled antibodies. In this experiment, an immunoassay with introduction of the product in a flow system is employed. The flow system acts as a linkage between the batch immunoassay and the detection system. The coupling of the flow system implies an important advantage when compared with batch protocols of immunoassays that use AP as label and detect the enzymatic product of 3-IP: (i) The electrode pretreatment commonly necessary for obtaining a reproducible methodology is simpler because the flow acts cleaning its surface; (ii) on the other hand, the accumulation step is reduced to the passage of the substrate loop by the electrode so that analysis time is decreased, and (iii) as the measurement is performed after the adsorption in fresh electrolyte, the influence of interfering species can be eliminated. However, AdSV and ACV could be demonstrated in a simpler way without requiring an immunoassay and a flow system as exemplified for other analytes such as melatonin [15]. This analyte (considered in Chapter 9) presents a reversible pair, whose current is increased after accumulation. It seems that the system can be a very interesting alternative and very appropriate also to study adsorption processes. The experiment combines immunoassay/flow/adsorptive stripping/ACV. Discussion of possible combinations with only two or three elements can be very interesting. 3-IP could be employed also as a substrate for other enzyme, also employed commonly in enzymatic immunoassays, horseradish peroxidase [16].
5.8 Assessment and discussion questions 1. Explain the basis of the electrochemical technique employed for the detection and suggest other possibilities. 2. What is the format of the immunoassay employed and what are the main steps? 3. Draw a scheme for the FIA system. 4. What is the difference between indigo blue and indigo carmine? 5. Explain the electrochemical behavior of indigo carmine. 6. Compare the analytical characteristics of this method with others found in the bibliography for the same or similar analytes, using other labels and methodologies. 7. Look for other electrochemical immunosensors that use alkaline phosphatase as label and discuss their differences.
References [1] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, second ed., John Wiley & Sons, New York, 2001. [2] J. Wang, Analytical Electrochemistry, second ed., Wiley-VCH, New York, 2000. [3] A.A. Kulkarni, I.S. Vaidya, Flow injection analysis: an overview, J. Crit. Rev. 2 (2015) 19e24. [4] M. Trojanowicz, K. Kołaci nska, Recent advances in flow injection analysis, Analyst 141 (2016) 2085e2139.
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[5] C. Kokkinos, A. Economou, M.I. Prodromidis, Electrochemical immunosensors: critical survey of different architectures and transduction strategies, TrAC Trends Anal. Chem. 79 (2016) 88e105. [6] F.S. Felix, L. Angnes, Electrochemical immunosensors e a powerful tool for analytical applications, Biosens. Bioelectron. 102 (2018) 470e478. [7] E.C. Rama, A. Costa-García, Screen-printed electrochemical immunosensors for the detection of cancer and cardiovascular biomarkers, Electroanalysis 28 (2016). [8] C. Fernández-Sánchez, M.B. González-García, A. Costa-García, 3-Indoxyl phosphate: an alkaline phosphatase substrate for enzyme immunoassays with voltammetric detection, Electroanalysis 10 (1998) 249e255. [9] C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Pneumolysin ELISA with adsorptive voltammetric detection of indigo in a flow system, Electroanalysis 13 (2001) 559e566. [10] M.J. Bengoechea Álvarez, C. Fernández Bobes, M.T. Fernández Abedul, A. Costa-García, Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system, Anal. Chim. Acta 442 (2001) 55e62. [11] M. Saraiva, A.O. Garra, The regulation of IL-10 production by immune cells, Nat. Rev. Immunol. 10 (2010) 170e181. [12] A.O. Garra, F.J. Barrat, A.G. Castro, A. Vicari, C. Hawrylowicz, Strategies for use of IL-10 or its antagonists in human disease, Immunol. Rev. 223 (2008) 114e131. [13] C. Fernández-Sánchez, A. Costa-García, Voltammetric studies of indigo adsorbed on pre-treated carbon paste electrodes, Electrochem. Comm. 2 (2000) 776e781. [14] M.J. Bengoechea Álvarez, M.T. Fernández Abedul, A. Costa García, Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes, Anal. Chim. Acta 462 (2002) 31e37. [15] J.L. Corujo-Antuña, S. Martínez-Montequín, M.T. Fernández-Abedul, A. Costa-García, Sensitive adsorptive stripping voltammetric methodologies for the determination of melatonin in biological fluids, Electroanalysis 15 (2003) 773e778. [16] P. Fanjul-Bolado, M.B. González-García, A. Costa-García, 3-Indoxyl phosphate as an electrochemical substrate for horseradish peroxidase, Electroanalysis 16 (2004) 988e993.
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