Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection

Figures

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  Figure A1. Cyclic voltammogram of SWCNT electrode with built-up immunosensor. Immunosensor on SWCNT electrode with 10 ng/mL of IL-13. Solution: 1 mM hydroquinone in 0.1 M PBS pH 6.0. Scan rate: 0.1 V/s.

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  Figure A2. Effect of the working potential on the immunosensor response determined in the beaker. Three different immunosensors prepared in the same manner (triplicates) for each data point. Amperometric measurements performed with 10 ng/mL of IL-13 (black circles) and with 0.0 ng/mL of IL-13 (white circles). Measurements in 10 mL of 1 mM hydroquinone in 0.1 M PBS pH 6.0 after addition of 100 μL of 50 mM hydrogen peroxide in 0.1 M PBS pH 6.0. Potentials of 0.0 V, −0.1 V, and −0.2 V were applied.

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  Figure A3. Raw data signals of IL-13 immunosensor measured in the beaker. Amperometric measurements performed at 0.0 V. Triplicates of sensors with 5.0 ng/mL of IL-13 (green) and with 1.0 ng/mL of IL-13 (black) prepared. Duplicates of sensor with 0.0 ng/mL of IL-13 (red) prepared. Measurements in 10 mL of 1 mM hydroquinone in 0.1 M PBS pH 6.0 after addition of 100 μL of 50 mM hydrogen peroxide in 0.1 M PBS pH 6.0.

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  Figure A4. Determination of flow parameters. Amperometric measurements performed at 0.0 V: (A) total flow 1 mL/min. For Q 1:10 (dashed line), continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.9 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6.0 with 0.1 mL/min. For Q 1:5 (solid line), continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.8 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6.0 with 0.2 mL/min. Injection volume 20, 50, and 100 μL; (B) total flow 1.2 mL/min. For Q 1:3 (dashed and solid line), continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.8 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6.0 with 0.4 mL/min. Injection volume 25, 50, 100, 125, 150, and 175 μL. For Q 1:3 (dotted line), continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.8 mL/min, injection flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.4 mL/min and injection volume 25, 50, and 100 μL.

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  Figure A5. Determination of temperature optimum of the IL-13 immunosensor in the flow cell. Amperometric measurements performed at 0.0 V. Sensor with 10 ng/mL of IL-13 (black circles) and sensor with 0.0 ng/mL of IL-13 (white circles), each measured two times in series at the same temperature. Temperature of 12 °C, 22 °C, 30 °C, and 37 °C applied. Measurements in flow cell with continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6 with 0.8 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6 with 0.4 mL/min, and injection volume of 100 μL.

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  Figure 1. Scheme of sensor set-up and signal generation. (A) Sensor set-up. Capture antibodies are covalently immobilized on a single-walled carbon nanotube (SWCNT) working electrode. Detection antibodies are biotinylated. The enzyme (violet) used for read-out is a streptavidin–horseradish peroxidase (HRP) conjugate. (B) Signal generation of the sensor. Reaction is initiated by adding hydrogen peroxide. Ox. = oxidized form. Red. = reduced form. Mediator (red.) = hydroquinone, mediator (ox.) = quinone. (C) Recorded signal. Change in current over time, when hydrogen peroxide is added.

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  Figure 2. Flow cell for temperature-controlled measurements. Flow cell mounted on aluminum block with fan. Two measurement compartments for dual measurement are available. Temperature control was realized by Peltier elements on the bottom of the cell with thermally conductive pads. In the lid of the cell: inlet, outlet, O-rings, and NTC thermistors. Electrical contacts of the fan and the Peltier elements are shown as well.

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  Figure 3. IL-13 ELISA as reference. Logarithmic representation of optical density vs. IL-13 concentration. For each concentration, duplicates were prepared according to the manufacture’s protocol. Read-out was performed at 450 nm and at 540 nm. Values at 540 nm were subtracted from values at 450 nm to correct for optical imperfections of the plate.

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  Figure 4. Characterization of the electrochemical IL-13 immunosensor in the beaker. Three different immunosensors prepared in the same manner (triplicates) for each concentration. Amperometric measurements performed at 0.0 V. Measurement in 10 mL of 1 mM hydroquinone in 0.1 M PBS pH 6 with 100 μL of 50 mM hydrogen peroxide in 0.1 M PBS pH 6 added to start read-out. Measurements at 25 ng/mL of IL-13 are not included in curve fitting.

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  Figure 5. Raw data signals of the electrochemical IL-13 immunosensor measured in the flow cell. Amperometric measurements performed at 0.0 V. Duplicates of sensor with 20 ng/mL of IL-13 (green), triplicates of sensor with 10 ng/mL of IL-13 (black) and with 0.0 ng/mL of IL-13 (red) prepared. Each sensor measured three times in series. Continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6.0 with 0.8 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6 with 0.4 mL/min and injection volume of 100 μL.

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  Figure 6. SEM images of SWCNT electrodes recorded at different excitation voltages and with different detectors: (A) resolution 1000×, excitation voltage 15 kV, SE detector; (B) zoomed in section of image (A), resolution 10,000×, excitation voltage 15 kV, SE InLens detector; (C) other electrode, resolution 100×, excitation voltage 5 kV, BSD detector, residues of organic solvents are visible as a dark shade.

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  Figure 7. Comparison of immunosensor characteristics in beaker and in flow cell. Three different immunosensors prepared in the same manner (triplicates) for each concentration. Amperometric measurements performed at 0.0 V. Measurements in beaker (black circles) in 10 mL of 1 mM hydroquinone in 0.1 M PBS pH 6, after addition of 100 μL of 50 mM hydrogen peroxide in 0.1 M PBS pH 6. Measurements in flow cell (white circles) with continuous flow of 1 mM hydroquinone in 0.1 M PBS pH 6 with 0.8 mL/min, injection flow of 1.5 mM hydrogen peroxide in 0.1 M PBS pH 6 with 0.4 mL/min and injection.