2 R ESULTS AND DISCUSSION
2.5 Uric acid sensing
In terms of application, the films were tested for the electrochemical detection of uric acid.
As an initial attempt, the redox behaviour of the Fe/L films deposited for 1 hour and for 15 minutes were studied by cyclic voltammetry, in phosphate-buffered saline (PBS) solutions. A total of 11 cycles were performed (Figure 2.49) and the films appeared stable. The current dif-ferences could be attributed to minimal film detaching from the electrode.
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.3
-0.2 -0.1 0.0 0.1
i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl) Fe/L 1h
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.2
-0.1 0.0 0.1
i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl) Fe/L 15 min
Figure 2.49 - Cyclic voltammograms of the 1-hour and 15-minute Fe/L films (2:1:1 ratio of M:H4L:PB, respec-tively) deposited on the FTO working electrode (0.7 cm2). CVs were performed in 10 mL of N2 saturated PBS buffer and recorded at 100 mV.s-1. The one-compartment electrochemical cell was also constituted by platinum
wire as counter and 3 M Ag/AgCl as reference electrodes.
The UA detection was further tested with differential pulse voltammetry (DPV) (Figure 2.50).
Concentrations of UA were added in a range of 300 to 550 μM and the current response was measured with a potential range between 0.4 and 1.1 V. The 15-minute Fe/L film showed a sensing tendency only between 300 and 350 μM of UA, by what seemed to have reached a limit. On the other hand, the 1-hour film did not show a tendency at all. The voltammograms started by showing an oxidation peak at 0.4 V by the addition of 300 μM of uric acid. When adding 350 μM, which was the second addition of uric acid, this peak unfolded into two peaks, at 0.4 and ~0.85 V and it was not possible to produce a calibration curve.
0.2 0.4 0.6 0.8 1.0 0.02
0.04 0.06
i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl)
300 M 350 M 400 M 450 M 500 M 550 M
Fe/L 1h
0.4 0.6 0.8 1.0
0.00 0.02 0.04
300 350 400 450 500 550 0
5 10 15 20 25 30 35 40
ip (uA.cm-2)
CUA (uM)
i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl)
300 M 350 M 400 M 450 M 500 M 550 M
Fe/L 15min
Figure 2.50 - DPV voltammograms of UA concentrations on a range of 300 to 550 μM on 1-hour and 15-minutes Fe/L films (2:1:1 ratio of M:H4L:PB, respectively) in PBS buffer.
Qualitatively, after the electrochemical sensing measurements, the dark-blue film turned white (Figure A.96 in Annexes). This proved that the films sensing abilities were being per-turbed by the simultaneous adsorption of a white component of the buffer, possibly the phos-phate anions. To overcome this limitation, the buffer solution was replaced by tris-HCl buffer.
The stability of the 1-hour and 15-minute Fe/L films in tris was analysed by performing CVs with 6 cycles (Figure 2.51). It was possible to observe a minor decrease of the current as the number of cycles increased, and that could also be associated with film detaching from the electrode.
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 -0.12
-0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10
Blank Fe/L 1h i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl)
-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 -0.10
-0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08
i (mA.cm-2 )
E (V vs. 3 M Ag/AgCl) Blank
Fe/L 15min
Figure 2.51 - Cyclic voltammograms of the 1-hour and 15-minute Fe/L films (2:1:1 ratio of M:H4L:PB, respec-tively) deposited on the FTO working electrode (0.7 cm2). CVs were performed in 10 mL of N2 saturated 0.1 M of
tris-HCl buffer and recorded at 100 mV.s-1. The one-compartment electrochemical cell was also constituted by platinum wire as counter and 3 M Ag/AgCl as reference electrodes.
The response of the films towards different concentrations of uric acid was once again meas-ured by DPV. Uric acid was added at concentrations between 5 and 325 μM and from the maximum peak currents, calibration curves were obtained.
The response of the unmodified FTO electrode was recorded to be compared with the Fe/L films (Figure 2.52). It resulted in a linear regression of ip = 0.0322 CUA + 4.033 (R2=0.9867) with a linear range of 85-325 μM.
0.0 0.2 0.4 0.6 0.8 1.0
4 6 8 10 12 14
100 150 200 250 300 350
6 8 10 12 14
ip (A.cm-2)
CUA (M) y = 0.0322x + 4.033 R2 = 0.9867
165 m UA 185 m UA 205 m UA 225 m UA 245 m UA 265 m UA 285 m UA 305 m UA 325 m UA
i ( A.cm
-2)
E (V vs. 3 M Ag/AgCl)
Blank 5 m UA 25 m UA 45 m UA 65 m UA 85 m UA 105 m UA 125 m UA 145 m UA
FTO
Figure 2.52 - DPV voltammograms of UA concentrations on a range of 50 to 325 mM on FTO electrode in tris buffer.
The response of the Fe/L film deposited for 1 hour on FTO was also measured (Figure 2.53).
It resulted in a linear regression of of ip = 0.01158 CUA + 28.01 (R2=0.989) with a linear range of 25-305 μM.
The response of the Fe/L film deposited for 15 minutes was also obtained by DPV (Figure A.97 in Annexes). This time, the peaks attributed to uric acid oxidation shifted to different potentials and did not show a current increasing tendency. The 15-minute film was far thinner than the 1-hour Fe/L film. Moreover, the 15-minute film had visible film holes, exposing the FTO electrode. The uric acid response irregularities could be influenced by the simultaneous response of the FTO electrode and so, the 15-minute film was not viable for the uric acid sens-ing studies.
0.3 0.4 0.5 0.6 0.7 0.8 0.9 28
30 32 34
0 50 100 150 200 250 300
28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0
ip (A.cm-2 )
CUA (M) y = 0.01153x + 28.313 R2 = 0.9892
i (A.cm-2 )
E (V vs. 3 M Ag/AgCl)
5 M UA 25 M UA 45 M UA 65 M UA 85 M UA 105 M UA 125 M UA 145 M UA
165 M UA 185 M UA 205 M UA 225 M UA 245 M UA 265 M UA 285 M UA 305 M UA 325 M UA
Fe/L 1h
Figure 2.53 - DPV voltammograms of UA concentrations on a range of 50 to 325 mM on 1-hour Fe/L film in tris buffer.
To conclude about the applicability of the Fe/L film, sensing parameters such as the limit of detection (LOD, calculated by Equation 7), sensitivity and standard deviation () were ob-tained and presented in Table 2.5.
𝐿𝑂𝐷 =3.3𝜎
𝑆 (Equation 7)
where (A.cm-2): standard deviation and 𝑆 (A.M-1.cm-2): slope of the curve.
As noticed, FTO electrode revealed a smaller linear range than the Fe/L film. In terms of sen-sitivity, they both display an approximately linear response to the quantity of uric acid, which is a positive result. Nevertheless, the Fe/L film similar sensitivity to the FTO. The standard deviation is higher for FTO. This means that the Fe/L film displayed a more accurate result and so, remains advantageous against bare FTO.
Table 2.5 - Sensing parameters obtained for Fe/L film deposited on FTO and for bare FTO.
Linear range (M) Sensitivity
(A.M-1.cm-2) (A.cm-2) LOD (mM)
Fe/L 25-305 0.012 1.037 0.296
FTO 85-325 0.032 2.523 0.259
As stated above in Chapter 1.1, normal uric acid levels range from 0.13-0.46 mM in blood and 1.49-4.5 mM in urine. Fe/L showed a LOD of 0.296 mM, which was slightly higher than the FTO. Moreover, it will not allow to quantify lower concentrations of uric acid in urine samples.
Comparing these results with the reported,[30] the sensor of this work presents an abnormally
high LOD, since the ones reported are produced with a LOD in a M scale and so, further sensor optimisations are needed. Due to time limitations, Fe/L1-L3 films were still not tested for uric acid sensing.