Characteristics of the MWCNT-PDMS electrodes
The peak currents were measured by differential pulse voltammetry (DPV) as a function of the weight ratio of the MWCNTs to PDMS in the MWCNT-PDMS electrodes; the peak current was observed to increase with increasing ratio of MWCNTs in the MWCNT-PDMS composite (Fig. 2A). In the case of 0.25:1, the DPV peak current was 0.8 µA at 0.72 V. When the MWCNT proportion was increased to 0.40:1, the peak current increased to 5.0 µA at 0.45 V; the main reason for this increase in peak current and decrease in peak potential with decreasing PDMS proportion can be due to the dielectric properties of PDMS37, which cause changes in the electron transfer kinetics between the electrode interface and electrolyte38,39. When the composite ratio was higher than 0.40:1, the composite was not of a paste form available for screen-printing.
(A) Differential pulse voltammograms of screen-printed MWCNT-PDMS electrodes with different mixing ratios of MWCNT and PDMS. (B) Peak currents measured for various concentrations of COOH-CNTs. The inset shows the differential pulse voltammograms for each deposition concentration of COOH-CNT on the working electrode. (C) Photograph of the working electrodes after placing 2 µl of dimethylformamide containing carboxyl-functionalized single-walled carbon nanotubes (COOH-CNTs) for different concentrations ranging from 0 to 1000 µg/ml, followed by curing in an oven at 100 °C. (D) Peak currents of the COOH-CNT/MWCNT-PDMS electrodes measured with repeating bending and unfolding for 20 cycles. The inset shows the photographs of the bending test setup. (E) Differential pulse voltammograms of the COOH-CNT/MWCNT-PDMS electrodes at 1, 5, 10, 15, and 20 bending cycles. (F) Peak currents measured for different antibody concentrations on the working electrodes (incubation time = 120 min, concentration: 1, 5, 10, 15, and 20 µg/ml). (G) Peak currents measured for antibody incubation times (concentration: 10 µg/ml, t = 0, 20, 40, 60, 80, 100, and 120 min, temperature = 37 °C).
Working electrode characterization and bending test
We examined the effects of the concentration of COOH-CNTs, and the concentration and incubation time of antibodies on the COOH-CNTs modified electrodes. Figure 2B shows the peak currents measured after immobilizing 2 μl of varying concentrations (0–1000 µg/ml) of COOH-CNTs on the working electrodes and baking them in an oven at 100 ℃. Increases in peak current and shifts in peak potential (from 0.45 to 0.2 V) were observed after immobilization of COOH-CNTs on the MWCNT-PDMS electrode. The reason for this can be attributed to high conductivity of COOH-CNT that enhanced the electron transfer between the working electrodes and electrolyte38,39, also implying that the electrochemical surface characteristics of the MWCNT-PDMS electrode changed because of COOH-CNT deposition. The peak current increased with increase in the COOH-CNTs concentration, and saturated at 18.25 μA for a concentration of 500 μg/ml, where a peak potential of 0.45 V was no longer observed. This means that COOH-CNTs sufficiently covered the PDMS-MWCNT electrodes in terms of electrochemical measurement.
For visual observation, images of the working electrodes were also captured with a smartphone camera after various concentrations of COOH-CNTs were immobilized on the working electrodes and dried in an oven at 100 ℃ (Fig. 2C). The bright gray color in the images indicate the deposition of COOH-CNTs on the electrode surfaces. The coffee ring effect40 was observed after deposition of COOH-CNTs in the concentration range from 50 to 200 µg/ml, where the black color showed negligible deposition of COOH-CNTs on the surface, demonstrating that these concentrations of COOH-CNTs were not enough to cover the whole electrode surfaces. However, the electrodes were fully covered (bright gray color) with COOH-CNTs after deposition of COOH-CNTs in a concentration range from 500 to 1000 µg/ml, which indicates that 500 µg/ml is the optimal concentration for complete coverage without clumps on the edges. This CNT concentration is comparable to that observed in another study, where the CNT concentration was around 200 μg/ml when using the drop-casting method41.
To assure the robustness and flexibility of these paper-based sensors, the peak currents of these electrodes were measured using DPV by repeatedly bending and unfolding twenty times (Fig. 2D)42. The peak currents varied from 18.8 μA to 20.4 μA, where the relative standard deviation (RSD) was 1.88%, which was lower than that (3.09%) observed in the reproducibility tests. This shows that the COOH-CNT/MWCNT-PDMS electrodes were minimally affected by mechanical bending, while the electrochemical sensors were continuously wetted, and the bent COOH-CNT/MWCNT-PDMS electrodes exhibited very good stability (Fig. 2E). Although the bending test is one of the major considerations for a flexible biosensor, paper-based electrochemical biosensors showing a bending test have rarely been presented to date42,43. This may be because such sensors are usually developed as disposable devices, and continuous monitoring of airborne viruses using disposable and flexible immunosensors, which may require that the sensors should be bent and unfolded21, has rarely been reported despite its high impact44,45. Moreover, the proposed MWCNT-PDMS electrodes on a paper substrate have the potential for flexible electrochemical biosensor applications, such as polymer-based roll-to-roll printed biosensor patches46.
The peak currents were also measured after immobilizing 10 μl of antibody solution on the modified MWCNT-PDMS electrodes in a concentration range from 1 to 20 μg/ml (Fig. 2F). The peak current decreased with increase in the concentration of antibodies up to 10 μg/ml, which might be due to insulating behavior of antibodies. When the antibody concentration was greater than 10 μg/ml, the peak current became saturated, which indicated that most of the free and activated carboxyl groups on the working electrodes bound with the antibodies. Optimization was also performed for incubation time of the antibodies. Ten microliters of antibodies in PBS (concentration: 10 μg/ml) was dropped on the modified MWCNT-PDMS electrodes and incubated in an oven at 37 ℃ up to 120 min to measure changes in the electrochemical properties with time (Fig. 2G). The peak current decreased with incubation time and converged at durations over 80 min.
Electrochemical characterization of the functionalized working electrodes
Electrochemical properties of the functionalized working electrodes with the deposition process were measured using DPV, CV, and EIS. Figure 3A shows the cyclic voltammograms for the MWCNT-PDMS (bare electrode), COOH-CNT/MWCNT-PDMS, Ab/COOH-CNT/MWCNT-PDMS, and BSA/Ab/COOH-CNT/MWCNT-PDMS electrodes. We observed a peak current of 38.9 μA at 0.2 V after deposition of COOH-CNTs on the bare electrodes, which had no CV peaks at 0.2 V. This was due to conductive behavior of COOH-CNTs via increasing the surface area and the electron transfer rate between the electrodes and electrolyte. The peak current decreased up to 29.9 µA after immobilizing antibodies on the modified electrode surface, which may be due to insulating behavior of antibodies. The peak current further decreased to 21.5 μA after immobilization of BSA. Large size and insulation properties of proteins such as BSA and antibodies decrease the electron transfer between electrodes and electrolyte11. DPV was also used for the same processes (Fig. 3B) and similar behaviors were observed. The peak currents were 16.9 μA after COOH-CNT deposition, 14.0 μA after antibody deposition, and 9.8 μA after BSA deposition.
(A) Cyclic voltammograms (scan rate: 50 mV/s), (B) differential pulse voltammograms, and (C) electrochemical impedance spectra of the MWCNT-PDMS, COOH-CNT/MWCNT-PDMS, H5N1-Ab/COOH-CNT/MWCNT-PDMS, and BSA/H5N1-Ab/COOH-CNT/MWCNT-PDMS electrodes. The inset shows the equivalent circuit for EIS measurement. (D) Measured values of the solution resistance (Rs), charge transfer resistance (Rct) and double layer capacitance (Cdl) in the MWCNT-PDMS, COOH-CNT/MWCNT-PDMS, H5N1-Ab/COOH-CNT/MWCNT-PDMS, and BSA/H5N1-Ab/COOH-CNT/MWCNT-PDMS electrodes. (E) The DPV peak currents measured for different incubation times for 10 µl of H5N1 HA detection (0, 10, 20, 30, and 40 min, concentration: 100 pg/ml, temperature = 25 °C). The error bars represent the standard deviations of three independent measurements.
The impedance spectroscopy was performed to measure the interfacial changes after each modification of the electrode surface (Fig. 3C) at an electric potential of 0.1 V and an amplitude of 10 mV in a frequency range from 0.1 Hz to 100 kHz. The inset shows the equivalent circuit, where Rct, Rs, Cdl and W represent the charge transfer resistance, solution resistance, double layer capacitance, and Warburg effect, respectively (Fig. 3D). The charge transfer resistance (Rct) of 35.5 kΩ for the bare electrodes was significantly reduced to 1.04 kΩ after COOH-CNT deposition. The Rct of the Ab/COOH-CNT/MWCNT-PDMS increased to 4.53 kΩ due to decrease in electron transfer after antibody immobilization, which hinders electron transfer. The Rct value of the BSA/Ab/COOH-CNT/MWCNT-PDMS electrodes were increased to 7.0 kΩ, indicating that the BSA covered nonspecific sites of the Ab/COOH-CNT/MWCNT-PDMS electrodes and obstructed the electron transfer.
The effect of incubation periods was measured to determine the optimal detection time for efficient binding of HA proteins on the antibodies-modified electrode surfaces. Figure 3E shows the peak currents after incubating H5N1 HA on the BSA/H5N1-Ab/COOH-CNT/MWCNT-PDMS electrodes for various time periods (10–40 min). Incubation of H5N1 HA on the sensors resulted in an antigen–antibody reaction and hence a change in the DPV peak current. The peak current saturated at 20–30 min, resulting in the maximum antigen–antibody binding.
Differential pulse voltammetry for hemagglutinin antigen detection
Three different HAs of the AI viruses (H5N1, H7N9, and H9N2) were detected using DPV (Fig. 4). Ten microliters of the HA solutions (in PBS) of different concentrations (10 pg/ml–100 ng/ml or 0.00017 nM–1.7 nM) were incubated on the BSA/Ab/COOH-CNT/MWCNT-PDMS electrodes for 30 min and then washed with PBS, followed by measurements with DPV. Figure 4A–C showed decreases in peak current with increases in concentration of HA proteins of H5N1, H7N9, and H9N2, respectively, in the range of 10 pg/ml–100 ng/ml. The previous studies for HA detection using electrochemical sensors were conducted in a similar concentration range (Table 1). The injected HA bound with the antibodies immobilized on the working electrodes, and peak currents decreased with increasing HA concentrations due to the electrical insulation property of HA proteins.
Differential pulse voltammograms of different concentrations of hemagglutinin antigens of the influenza virus (A) H5N1, (B) H7N9, and (C) H9N2 subtypes. (D) Calibration graphs of hemagglutinin antigens of influenza virus H5N1, H7N9, and H9N2 for the detection range from 100 pg/ml to 100 ng/ml. The error bars represent the standard deviations of three independent measurements.
Figure 4D shows the log-linear relationships between the DPV peak currents and the three HA (H5N1, H7N9, and H9N2) concentrations. The LODs were computed as 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA, based on the three signal-to-noise ratios. Compared to other works on electrochemical detection of influenza HAs (Table 1), where the LODs were 9.4 pM15 and 1 pM16 for H5N1 HA proteins, present sensors showed similar or enhanced detection limits for H5N1 HA proteins. Han et al.33 reported a microfluidic immunosensor for simultaneous detection of H1N1, H5N1, and H7N9 viruses using ZnO nano-rods and HRP conjugated antibodies for signal enhancements with LOD of 1 pg/ml; however, the detection time was long (more than 2 h) because they used 1 h for incubation time of antigens and then another 1 h incubation of conjugated antibodies, followed by washing and detection33. The present sensors showed rapid (20–30 min) detection of multiple AI antigens using a simple, cost-effective and flexible paper-based electrochemical immunosensor. Moreover, there are no other studies reported on multiple detection of H5N1, H7N9, and H9N2 HA antigens in a single electrochemical paper-based device.
Multiple detection test
To demonstrate the detection capabilities of the sensors for multiple targets, we tested one case in this study although there are many combinations of 3 three different HAs and several antigen concentrations. A 10-μl PBS containing H5N1 HA (10 ng/ml) and H9N2 HA (10 ng/ml), where no H7N9 HAs were included, was injected into each of the three working electrodes (BSA/H5N1-Ab/COOH-CNT/MWCNT-PDMS, BSA/H7N9-Ab/COOH-CNT/MWCNT-PDMS, and BSA/H9N2-Ab/COOH-CNT/MWCNT-PDMS electrodes) and incubated for 30 min at room temperature before measuring the electrochemical changes on each working electrode sequentially (Fig. 5A,B). The peak currents measured after the HA sample injection were 6.6 μA and 6.9 μA for H5N1-Ab and H9N2-Ab functionalized electrodes, respectively, whereas 11.9 μA was obtained for H7N9-Ab functionalized electrodes, similar to those of the negative controls. The peak currents of the single HA tests showed 5.8 μA (p = 0.19) at 10 ng/ml of H5N1 HA and 7.6 μA (p = 0.84) at 10 ng/ml of H9N2 HA (Fig. 4D), demonstrating that both single and multiple analyte detections were not significantly different for the 95% confidence intervals. Based on the observations, these immunosensors can quantify the concentrations of multiple HA proteins and showed negligible interference between target and non-target HAs in the samples.
(A) Differential pulse voltammograms and (B) peak currents of three BSA/Ab/COOH-CNT/CNT-PDMS electrodes of the proposed immunosensors after 10 (mathrm{mu l}) of 1 × PBS containing 10 ng/ml of H5N1 HA and 10 ng/ml of H9N2 HA was injected and incubated at room temperature for 30 min. (C) Selectivity of the immunosensors (H9N2-Ab functionalized electrodes) for influenza virus H9N2 hemagglutinin antigen detection. The test included 1 × PBS only, ten-fold diluted human serum only, non-targets (H5N1 HA, H1N1 virus, and MS2 bacteriophage) in 1 × PBS, and targets in 1 × PBS or ten-fold diluted human serum. (D) Reproducibility of the immunosensors (H5N1-Ab functionalized electrodes) using H5N1 HA antigen (concentration: 100 ng/ml). The error bars represent the standard deviations of three independent measurements.
Selectivity and reproducibility tests
For selectivity assay, different viruses (influenza A H1N1 whole viruses and MS2 bacteriophages) and HA proteins in PBS and human serum were used (Fig. 5C). The human serum was ten-fold diluted with PBS. H9N2-Ab functionalized electrodes were incubated with 10 μl of either H5N1 HA, H1N1 viruses, H9N2 HA (target), or MS2 bacteriophages for 30 min and washed with PBS before DPV measurements. The negative controls (PBS and human serum) were obtained by measuring the peak current of the H9N2-Ab functionalized electrodes after incubation of PBS and human serum alone, respectively. The concentration used in the experiments was 10 ng/ml for HA and 104 plaque forming units (pfu)/ml for viruses and bacteriophages. Only the DPV peak currents for H9N2 HA decreased, unlike those for the other non-targets and the negative controls. This implies that the immunosensor shows good selectivity.
The reproducibility tests of these sensors were conducted (Fig. 5D). All H5N1-Ab functionalized electrodes were given the same concentration of H5N1 HA (100 ng/ml), incubated for 30 min, washed with PBS, and measured by DPV. The peak currents did not differ significantly, and the RSD was 3.09%, showing good reproducibility for label-free detection.
