Electrochemical detection of uric acid in undiluted human saliva using uricase paper integrated electrodes

Abstract

In this study, we introduce a uricase-immobilized paper (UOx--paper) integrated electrochemical sensor for detection of uric acid (UA) in saliva. The UOx was immobilized on the detection zone in the wax-patterned paper substrate. This UOx-paper was integrated with a Prussian blue--modified, screen-printed carbon electrode after electropolymerization of o-phenylenediamine to construct an electrochemical cell for small-volume (20 μL) of samples. First, we optimized the fabrication conditions of UOx-paper. Next, the amperometric response of the UOx-paper-based electrochemical UA sensor was analyzed using a known concentration of UA standard solution in artificial saliva at an applied potential of - 0.1 V (versus Ag pseudo-reference electrode). The UOx--paper based electrochemical UA sensor showed a sensitivity of 4.9 μA·mM^-1 in a linear range of 50 to 1000 μM (R² = 0.998), high selectivity and good reproducibility, as well as a limit of detection of 18.7 μM (0.31 mg/dL) UA. Finally, we quantified the UA levels in undiluted saliva samples of healthy controls (n = 20) and gout patients (n = 8). The levels were correlated with those measured with conventional salivary UA enzymatic assays as well as serum UA levels. The UOx-paper-based electrochemical UA sensor is a user-friendly and convenient tool to assess salivary UA levels.

Figure 1

Schematic representation of the sensing principle and resulting current signal of the UOx-paper-based electrochemical UA sensor, and uric acid concentrations (mg/dL) in human saliva samples.

Figure 2

(a) Cyclic voltammograms of PPD/PrB-SPCE in an artificial saliva solution with and without 1.0 mM H₂O₂ at a potential ranging from − 0.2 to 0.4 V (vs. Ag pseudo-reference electrode) and a scan rate of 50 mV/sec. (b) Current response to 0, 50, 100, and 200 μM H₂O₂ of PPD/PrB-SPCE compared with response to common electroactive interferences including 1000 μM UA, 500 μM AA, and 500 μM AP. (c) Comparison of the electrochemical response of PPD/PrB-SPCE and PrB-SPCE to 50 μM H₂O₂ and 1000 μM UA, and 500 μM AA, and 500 μM AP, respectively, at − 0.1 V (vs. Ag pseudo-reference electrode). Effect of (d) UOx concentration, (e) drop volume of UOx mixture, and (f) UOx immobilization method on the analysis of 1000 μM UA. Error bars represent standard deviation of the mean (n = 3).

Figure 3

(a) The calibration curve of the cathodic current vs. UA concentration in the comparison of UOx-paper/PPD/PrB-SPCE and UOx-membrane/PPD/PrB-SPCE at − 0.1 V (vs. Ag pseudo-reference electrode), respectively. (b) The current response to 500 μM UA of UOx-paper/PPD/PrB-SPCE at − 0.1 V (vs. Ag pseudo-reference electrode) compared with response to common electroactive physiological interferences including 800 μM glucose, 200 μM AA, 100 μM AP and 1000 μM LA. (c) Reproducibility of UOx-paper/PPD/PrB-SPCE with different fabrication dates, based on current response to 500 μM UA in artificial saliva at − 0.1 V (vs. Ag pseudo-reference electrode). (d) Stability of UOx-paper/PPD/PrB-SPCE based on the current response to 500 μM UA stored for 28 days at 4 °C. Error bars represent standard deviation of the mean (n = 3).

Figure 4

Uric acid (UA) levels in serum and unstimulated whole saliva samples. (a) Patients with gout (n = 8) showed significantly higher levels of serum or salivary UA than control subjects (n = 20, all p < 0.05). Salivary UA levels measured with our UA sensor were significantly lower than serum and salivary UA levels measured via conventional enzymatic colorimetric assays in both controls and gout cases (both p < 0.0001 by ANOVA). Error bars represent standard error of mean. (b) UA levels in serum or saliva (measured with Salimetrics® and UA sensor) were significantly positively correlated with each other.

Figure 5

Change in uric acid (UA) levels in serum and unstimulated whole saliva samples after urate-lowering therapy. (a) In patients with gout (n = 8), serum UA levels decreased significantly after the treatment. However, salivary UA levels were not significantly changed regardless of the method used. (b) Changes in serum UA were significantly correlated with changes in salivary UA levels measured with our UA sensor (p < 0.0001), but not Salimetrics® assay.