Development of albumin detection using a redox cycling process coupled with a paper-based device

Abstract

Albumin is an important indicator of albuminuria, which is related to a risk of chronic kidney disease (CKD). High albumin levels in urine indicate the onset of diabetic nephropathy, which can progress to irreversible renal dysfunction if patients are not medicated. This situation could result in premature death, a lower quality of life, and high healthcare costs. Every year, the number of patients with CKD increases. For example, the previous report in 2020 {Yang, 2020 #65}[1] indicated that the prevalence of CKD at the 3^rd and 4^th stages in the Thai population over the age of 18 was estimated to be 9.3%, representing 4.8 million patients, whereas 4.6 million Thai people also suffer from CKD at the 1^st and 2^nd stages. Thus, urinary albumin measurement is highly required for screening a person who is at risk of kidney disease and preventing the progression of this serious health issue. The major challenge of albumin detection is selecting an accurate and sensitive analytical method to detect albumin simply and rapidly to support a 24-h urine collection, which is the “gold standard” method in clinical testing. The electrochemical method is one of is the solutions to this challenge because of its high sensitivity, low-cost instrument, portability, and ease of operation. The majority of previous research suggests that using nanomaterials and molecularly imprinted polymers (MIPs) for electrode modification can improve sensitivity and selectivity [2]. However, the use of large-scale conventional electrode systems and the complicated modification steps are the main problems with those methods, limiting their applications for rapid albumin screening in developing countries. As a result, the strategy of improving sensitivity by using unmodified electrodes promptly motivated interest in overcoming these drawbacks. In this work, we proposed an electrochemical-chemical (EC) redox cycling process to amplify the signal by using a mixture solution of ferricyanide ([Fe(CN)₆]^3-) and methylene blue (MB), two commonly used electroactive species in biosensors. These solutions were prepared in the same tube with phosphate buffered-saline solution (PBS) at pH 7.4 as a supporting electrolyte and labeled as in the absence of albumin. In the presence of albumin, a redox cycling solution and bovine serum albumin (BSA) standard or urinary sample solution were mixed in the same tube. For detection, 100 µL of a pure redox cycling solution or that mixed with albumin or a sample solution was thoroughly dropped onto an electrochemical paper-based analytical device (ePAD), which was fabricated from paper as a substrate. Subsequently, electrochemical techniques, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were performed. The detection procedure can be completed in 1 min, as presented in Fig 1. In this study, albumin was quantified by subtracting the signal current of a redox cycling solution without BSA standard or urinary sample from the signal current of a redox cycling solution with BSA standard or urinary sample. For the results, we strongly believe that the EC redox cycling process can improve electron transfer efficiency across the interface in the absence of albumin. [Fe(CN)₆]^3- is first reduced to [Fe(CN)₆]^4- via an electrochemical reaction at the electrode surface, which is then re-oxidized back to [Fe(CN)₆]^3- by MB via a chemical reaction. Due to the simultaneous regeneration of [Fe(CN)₆]^3-, this redox cycling process continuously occurs, resulting in a large electrochemical signal. In contrast, when albumin was present in the redox cycling solution, the signal current obviously decreased. We hypothesized that this behavior was caused by negatively charged BSA blocking the electrode surface, preventing [Fe(CN)₆]^3- from accepting electrons from a working electrode. This circumstance resulted in a diminishing response because [Fe(CN)₆]^3- is hardly reduced to [Fe(CN)₆]^4-, which is then slightly re-oxidized by MB. This is the first study to use the EC redox cycling process for albumin detection coupled with a paper-based device. We believe that our assay can be used for a 24-h urine collection to accurately detect albumin in the urine of patients who are at risks of kidney diseases, possibly preventing the progression of CKD and reducing the number of severe patients. Furthermore, the EC redox cycling process provides a promising system for point-of-care testing because both [Fe(CN)₆]^3- and MB are inexpensive and available in general chemical laboratories, allowing them to support other biomolecule measurements for simple and highly sensitive detection.