Uric acid enzyme biosensor based on a screen-printed electrode coated with Prussian blue and modified with chitosan-graphene composite cryogel

Highlights

• Uricase/Chi-Gr cry/PB/SPCE is proposed for UA detection.

• The biosensor exhibits a low detection limit of 2.5 µmol L⁻¹.

• The Chi-Gr cryogel provided a large surface area and shows good stability.

• The biosensor can detect UA in human blood serum samples with high accuracy.

Abstract

An amperometric uric acid (UA) biosensor was developed by immobilizing uricase on porous cryogel (cry) platform of graphene-incorporated chitosan (Chi) on top of a Prussian blue layer (PB) electrodeposited on a screen-printed carbon electrode (Uricase/Chi-Gr cry/PB/SPCE). Amperometric detection of UA catalyzed by uricase was based on the change in cathodic current of PB at a potential of 0.00 V in a flow injection system. The UA biosensor showed a linear range between 0.0025 and 0.40 mmol L⁻¹ with a detection limit of 2.5 µmol L⁻¹ (S/N = 3). In addition, the Uricase/Chi-Gr cry/PB/SPCE provided an excellent stability that enabled reuse up to 175 times (RSD of 3.7%) and a good electrode-to-electrode repeatability (RSDs of 0.53–2.7%, n = 6). A Michaelis-Menten constant ($K_M^{app}$) of 0.23 mmol L⁻¹ indicated the excellent affinity of the immobilized uricase toward UA. The modified electrode showed no effect from the common interferences in human serum samples. When applied to measure UA in human serum samples, the developed UA biosensor achieved recoveries ranging from 98 ± 2 to 102 ± 5% and the results obtained were in good agreement with enzymatic colorimetric analysis (P > 0.05).

Introduction

Uric acid (UA) is a purine metabolism product used in clinical diagnosis. For healthy individuals, the normal concentration of UA is between 0.24 and 0.52 mmol L⁻¹ in blood serum and from 1.40 to 4.40 mmol L⁻¹ in excreted urine [1]. An abnormal UA level is a marker of gout [2,3], renal disease [4] and other disorders. Clinically, UA is commonly determined by an enzymatic colorimetric method, using uricase and peroxidase together, involving the catalytic oxidation of UA by uricase to allantoin, hydrogen peroxide and CO₂. After that, peroxidase can catalyze the hydrogen peroxide in the presence an oxygen acceptor to produce a colored product [5]. Although this method is highly specific and sensitive, the cost of uricase and peroxidase enzymes is expensive and the enzymes cannot be reused [6]. An interesting method of detecting UA is the use of an electrochemical uricase enzyme electrode. Simple, easy to miniaturize and highly selective, this type of electrode can detect the analyte at the submicro level using a small amount of enzyme that can be reused [2,7].

To determine UA levels, electrochemical biosensors are commonly used in an amperometric system that measures H₂O₂ produced during the uricase-catalyzed oxidation of UA [8]. The main issue with this approach is that H₂O₂ oxidation requires a high potential that also oxidized other electroactive species in clinical samples, including ascorbic acid and dopamine, resulting in interfering signals [9]. Strategies employed to lower the oxidation potential have included the use of horseradish peroxidase [10] or redox mediators [11], and some aspects of this type of biosensor have been improved with Prussian blue (PB), a mediator considered to be an “artificial peroxidase” [12]. Despite these improvements, advances could still be made in other directions, particularly in the extension of biosensor stability [8].

One of the determining factors of biosensor stability and reusability is the number of available active biosensing sites. Such sites can be immobilized on a supporting material [13,14], and the supporting platform chosen should preferably have a large surface area. Cryogel (cry), a macroporous material easily prepared by freezing and thawing [15], is a suitable choice because it provides a large surface area. The stabilization of the enzyme can also be improved by functionalization of the supporting material. The biocompatible polymer chitosan (Chi) has amino groups in its structure that enabled enzyme immobilization by crosslinking [15,16]. However, Chi is a non-conducting material, so electron transfer had to be accelerated by incorporating nanomaterials with good electrical conductivity, such as graphene (Gr) [17,18].

Therefore, this work reports for the first time the development and characterization of a UA biosensor that combines the materials mentioned above (Uricase/Chi-Gr cry/PB/SPCE). A porous Chi-Gr cry was used as the supporting material to immobilize uricase on an electrodeposited layer of PB. The analytical performances of the fabricated UA biosensor were characterized, including the linear concentration range, LOD, $K_M^{app}$, selectivity, and reproducibility. The biosensor was then applied to measure UA in clinical samples.