Highly permselective uric acid detection using kidney cell membrane–functionalized enzymatic biosensors
Introduction
Uric acid (UA) is the final decomposition product from the metabolism of purine, a constituent of nucleic acids, in the liver and is present in meat products. The blood UA level is regulated by the nephrological system, which excretes UA through the kidney. High UA (~350 μM) level could lead to gout, a condition characterized with the precipitation of needle-like UA crystals in the joints, skin, and other tissues, that is common in patients with diabetes, Lesch–Nyhan disease, Down syndrome, and Von Gierke disease (Abou-Elela, 2017). The incidence of gout has increased in recent decades (Singh et al., 2019).
Identification of UA crystals is imperative for the diagnosis of gout. At present, two methods, polarized light microscopy and X-ray imaging, are employed to detect UA crystals. UA crystals can be visually detected from the synovial fluid via polarized light microscopy, but sampling of the synovial fluid can be painful. X-ray imaging, on the other hand, allows non-invasive observation of UA crystals. These methods are expensive and require large equipment and some specialists. Furthermore, UA crystals can be detected only at the acute stage of gout, which is associated with painful intercritical segments from excessive UA crystal deposition in the joints. Thus, it is difficult to have an early diagnosis of gout using conventional methods (Vaidya et al., 2018).
To overcome these limitations, UA biosensors have been developed. Hyperuricemia, an early symptom of gout, can be detected using UA biosensors. Electrochemical UA detection has various advantages, such as early diagnosis, rapid measurement, high sensitivity, portable size, and ease of use (Mathew et al., 2021). Therefore, electrochemical enzymatic biosensors for sensitive UA detection have been constructed using nanomaterials composed of copper oxide, graphene oxide, and zinc oxide (Krishnan et al., 2019).
Despite their superior sensitivity, electrochemical UA biosensors are often easily interfered by antioxidants in the blood, such as lipoic acid, glutathione, and vitamins. Therefore, it is important to improve the selectivity of electrochemical UA biosensors for accurate UA detection. UA is selectively reabsorbed through uric acid transporter1 (URAT1) in the kidney to regulate its excretion. URAT1 is localized in the apical membrane of the kidney proximal tubular cells. It has been reported that cell membranes and membrane proteins can be functionalized onto various nanoparticles and solid surfaces to provide various functionalities, such as the selective transportation of molecules through the membrane (Stiburkova et al., 2019). Applying a URAT1-rich kidney cell membrane (KCM) on UA biosensors could potentially increase the selectivity of UA against antioxidants that cannot permeate the KCM (Kim et al., 2018).
In this study, we fabricated a uric acid oxidase (UOx)-immobilized sensor (called the UOx sensor) and a KCM-coated UOx sensor (called the KCM sensor). The surface morphology of the KCM sensor was verified via scanning electron microscopy (SEM), atomic force microscopy (AFM), and confocal microscopy, and its electrochemical properties were studied using cyclic voltammetry (CV). The sensing performance of the KCM sensor was confirmed to be in the range of 0–1000 μM UA. Its selectivity was demonstrated by testing five antioxidants in human plasma (Shindyapina et al., 2017). Finally, the feasibility, stability, and reproducibility of the KCM sensor were verified by evaluating its practical applications. Taken together, we demonstrate the development of a kidney-mimetic electrochemical biosensor functionalized with URAT1 to improve the accuracy of UA detection.
Section snippets
Materials and reagents
Screen-printed carbon electrodes (SPCEs, DRP–110) were purchased from DropSens. Nafion 117 (Naf), ferrocene (Fc), potassium ferricyanide ([Fe(CN)6]3−), potassium ferrocyanide ([Fe(CN)6]4−), potassium chloride (KCl), UOx, glutaraldehyde, ascorbic acid (AA), barbituric acid (BA), lipoic acid (LA), glutathione (GSH), niacinamide (NA), protease and phosphatase inhibitor cocktail, fetal bovine serum (FBS), bovine serum albumin (BSA), and penicillin–streptomycin G were purchased from Sigma-Aldrich.
Fabrication of KCM sensor
The working electrode of the SPCE was serially functionalized with the Fc-Naf and UOx mixture. Fc is widely used as an electronic mediator because it can promote stable electron transfer. To detect UA, the following chemical reactions occurred:
Equations (1), (2), (3) represent electrochemical oxidation, Fc oxidation, and electrochemical reduction, respectively.
KCM was diluted in PBS and sonicated for 5 min to produce KCM vesicles. The
Conclusion
We developed a highly selective UA biosensor using URAT1-rich KCM as a permselective filter. The KCM coating on the enzymatic sensor was verified through surface visualization and topological analysis. It was confirmed, by comparing the KCM and DOPC sensors, that URAT1 is a key factor in transporting UA to UOx immobilized on the sensor electrode. Furthermore, the UA permeation capability of URAT1 was demonstrated through its inhibition. The KCM sensor was permselective to UA and was unaffected
CRediT authorship contribution statement
Insu Kim: Investigation, Conceptualization, Methodology, Writing, Formal analysis, Writing – original draft, Cell membrane extraction. Young Im Kim: Investigation, Methodology, Visualization, Writing – review & editing. Sang Won Lee: Cell culture. Hyo Gi Jung: Visualization. Gyudo Lee: Writing – review & editing. Dae Sung Yoon: Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No., NRF-2018M3C1B7020722, NRF-2019R1A2B5B01070617, and 2020R1A6A3A01096477). This work was supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT), Project Number: 202012D19. This study was also supported by the BK21 FOUR (Fostering Outstanding Universities for Research).
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These authors contributed equally to this manuscript.