*3.7. Interference Studies*

One of the essential features of the sensor is its selectivity. CV measurements of a 10 μM UA solution in 0.1 M PB at pH 6 were conducted in the presence of common interferents found in biological matrices. Both organic and inorganic interfering species which exist in the lysed cell culture were taken into account. The measurements are performed in the potential range from −0.5 V to 1 V, at the scan rate of 50 mV/s, with the ratio of UA and potential organic interferents 1:1. The chemical behavior of UA in the presence of ascorbic acid (AA), citric acid (CA), dopamine (DOPA), gallic acid (GA) and glucose (GLU) is shown in the Supplementary Data (Figure S5A). While CA and GLU do not influence UA detection, the other interferents lead to signal elevation. However, the peak originating from UA remains visible and measurable in the presence of DOPA, AA and GA (Figure S5A). Although considerable interferences originating from the substances commonly present in the biological matrix present the major drawback of the proposed method, they can be overcome by careful experiment planning and execution and/or the use of chemometric methods. We proved this by measuring only the height of the peak originating from UA oxidation, which remains nearly the same in all samples, with the highest deviation for the ascorbic acid (histogram on Figure S5B).

Considering the fact that high concentrations of salts can be present in the sample, the influence of 0.1 M KCl, NaCl, CaCl2, MgOAc and NaOAc in 0.1 M PB at pH 6 on detection of 10 mM UA was studied. The results show that none of the mentioned salts in a 1000:1 ratio to UA influence UA detection. The interference study proves that the proposed sensing platform shows satisfying selectivity towards UA in the presence of common organic and inorganic interferents.

The practicability estimation of the developed method after the cell stress.

To analyse the applicability of the proposed sensing platform in biological samples, measurements were conducted in a real sample matrix. CV measurements were performed in the potential range from −0.5 V to 1 V at the scan rate of 50 mV/s in the presence and absence of UA to scan for potential interferents (Figure S6A). The baseline in the biological matrix gives a high current response, originating from the oxidation of electrochemically active species contained in the matrix. However, this does not influence the UA amperometric detection, because, at 0.3 V, where uric acid oxidation occurs, no matrix interferences are present. To prove this, a calibration curve is constructed once again in the biological sample matrix (DMEM). The obtained results are displayed by the amperometric curve (Figure S5B) and corresponding plot (inset Figure S6B). The amperometric current response changes linearly with the UA concentration in the range from 1 μM to 38 μM, which is described by the equation I (nA) = 4.1455 + 0.4326c μM, with the linear regression coefficient R = 0.9910.

After the calibration, real sample analysis was conducted. The samples were obtained by treating the human HEK293 and HeLa cell cultures with the papain, and the actinidin allergen, respectively, during a time frame of 12 h. Ten microliters of the sample was added to 5 mL of 0.1 PB at pH 6, and the change in the amperometric current at 0.3 V was measured. However, the UA concentration in the sample was found to be lower than the limit of quantification. Therefore, the sample was spiked with UA standard solution, so the final concentration of UA (spiked) was 100 μM. The measurements were repeated five times with the spiked sample, and the mean value of the rise in the amperometric signal corresponded to the 0.2010 mM UA concentration in the 5 mL of PB, which agrees with the total UA concentration in the sample of 100.5 μM. That proves that the proposed method is applicable in real sample analysis. However, it requires either more concentrated samples, larger quantities of samples, or spiking the samples with a known amount of UA.

To further support this claim, a series of HeLa and Hek293 cell cultures were treated with papain and EG4 inhibited papain for prolonged periods. The cell cultures were prepared in duplicate with each of the abovementioned allergens independently and the amperometric response at 0.3 V was recorded immediately after the addition of allergens, and after incubation periods of 3 h, 6 h and 12 h. The obtained results are given as mean values of the measurements in Table 2. The control groups of cells, without the added allergens, were also analyzed to compare the UA release from the treated and the untreated cells.

**Table 2.** The concentrations of UA obtained using the developed amperometric method (sensor) and HPLC as the reference method after the incubation of HeLa and HEK293 cells with papain.


The acquired values indicate that the stress on the cells after the adequately long (6 h and 12 h) exposure to papain is sufficient to release considerable amounts of UA. Furthermore, the quantity of UA released is directly proportional to the incubation period, proving that the developed method is suitable for real-time detection or monitoring of cell damage or stress.

For method validation, the same real samples were analyzed by using HPLC as a standard method. The obtained results display good agreement with those achieved in the developed electrochemical method. The recoveries of the determination are 94.3–103.0%, indicating good accuracy of the developed method in real sample analysis.
