Highly permselective uric acid detection using kidney cell membrane–functionalized enzymatic biosensors

https://doi.org/10.1016/j.bios.2021.113411Get rights and content

Highlights

  • Sensitive and selective uric acid detection platform was developed by functionalizing kidney cell membrane on uric acid sensor (KCM sensor).

  • URAT1 on kidney cell membrane allows uric acid to selectively permeate the cell membrane.

  • KCM sensor was able to detect uric acid without interference of antioxidants in human blood.

  • KCM sensor was stable for 3 weeks storage and highly reproducible (CV < 0.19 μA).

  • Human serum uric acid ranged from 35 to 835 μM was linearly detected (R2 = 0.9839).

Abstract

Abnormal blood uric acid (UA) levels can lead to its crystallization in the joints, consequently resulting in gout. Accurate detection of UA in the blood is imperative for the early diagnosis of gout. However, electrochemical UA biosensors are vulnerable to antioxidants in the blood, limiting accurate UA detection. To address this issue, we focused on the function of uric acid transporter 1 (URAT1), which is selectively permeable to UA. URAT1 is abundant in the kidney cell membrane (KCM). To apply URAT1 to a sensor, we developed a KCM-coated UA biosensor (called the KCM sensor) that could selectively detect UA through URAT1. The KCM coating in the fabricated KCM sensor was verified via scanning electron microscopy, atomic force microscopy, and confocal microscopy. The KCM sensor enabled the detection of UA in the range of 0–1000 μM, with a limit of detection of 8.5 μM, suggesting that it allows the diagnosis of the early stages of gout. On the other hand, the UA permeability of the KCM sensor was significantly reduced in the presence of a URAT1 inhibitor, implying that URAT1 is a key factor for UA detection. The selectivity of the KCM sensor was demonstrated by measuring the amount for UA in the presence of various antioxidants. Finally, the KCM sensor was capable of measuring UA in human serum and was reproducible with 0.5–1.6% deviation. The UA permeability and selectivity of the KCM sensor were maintained even after 3 weeks of storage.

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:FceFc+Uricacid+O2UOxAllatoin+CO2+2H++2eFc++eFc

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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