An Optical Urate Biosensor Based on Urate Oxidase and Long-Lifetime Metalloporphyrins

Abstract

Gout is a condition that affects over 8 million Americans. This condition is characterized by severe pain, and in more advanced cases, bone erosion and joint destruction. This study explores the fabrication and characterization of an optical, enzymatic urate biosensor for gout management, and the optimization of the biosensor response through the tuning of hydrogel matrix properties. Sensors were fabricated through the co-immobilization of oxygen-quenched phosphorescent probes with an oxidoreductase within a biocompatible copolymer hydrogel matrix. Characterization of the spectral properties and hydrogel swelling was conducted, as well as evaluation of the response sensitivity and long-term stability of the urate biosensor. The findings indicate that increased acrylamide concentration improved the biosensor response by yielding an increased sensitivity and reduced lower limit of detection. However, the repeatability and stability tests highlighted some possible areas of improvement, with a consistent response drift observed during repeatability testing and a reduction in response seen after long-term storage tests. Overall, this study demonstrates the potential of an on-demand, patient-friendly gout management tool, while paving the way for a future multi-analyte biosensor based on this sensing platform.

Keywords: gout; metalloporphyrins; optical biosensors; phosphorescence; urate.

Figure 1

Schematic of flow-through system for in vitro biosensor assessments of oxygen response.

Figure 2

Illustration of the urate biosensing mechanism based on enzyme-induced oxygen depletion coupled with oxygen-responsive phosphors immobilized in a biocompatible hydrogel matrix. Urate concentration is proportional to phosphorescence intensity and lifetime.

Figure 3

(A) Absorbance and (B) emission spectra of urate biosensor containing palladium (II) tetramethacrylated benzoporphyrin (BMAP) and uricase.

Figure 4

Effect of oxygen concentration on phosphorescence lifetime. (A) Raw data illustrating the changes in phosphorescence lifetime over time as oxygen concentration is reduced [n = 3 samples, 50:50 poly(2-hydroxyethyl methacrylate (HEMA)-co-acrylamide (AAm))]. These data are representative of a typical oxygen response profile obtained from a single oxygen response test. (B) Oxygen response of the three hydrogel urate biosensor compositions. Each data point represents a steady-state average of n = 3 samples of the sample composition. Error bars represent 95% confidence intervals.

Figure 5

Effect of urate concentration on phosphorescence lifetime. (A) Raw data illustrating the change in phosphorescence lifetime over time with increases in urate concentration. Data are representative of a typical urate response profile obtained from a single test [n = 3 samples, 50:50 poly(HEMA-co-AAm)]. (B) Urate responses obtained from the three different hydrogel urate biosensor compositions. Each data point represents a steady-state average of n = 3 samples of the sample composition. Error bars represent 95% confidence intervals.

Figure 6

Response of urate biosensors stored in 5 mg/dL urate solution dissolved in 10 mM PBS. Both solutions were stored at 25 °C and pH 7.4 over an eight-week period. Measurements were made at the beginning, middle, and end points of the eight-week period. Error bars represent 95% confidence intervals. A single asterisk indicates a p value of 0.05 or less, while a double asterisk denotes a p value of 0.01 or less.