Robust Electrochemical Sensing Techniques for Serum Creatinine Biosensor
Creatinine is an important biomarker for evaluating the renal function and its concentration in serum can be utilized for early detection of kidney disease, thyroid disorders, and muscular dystrophy. Accurate, reliable, and decentralized testing of creatinine has become vital owing to the rising trajectory of Chronic Kidney Disease and its associated risk factors – diabetes and hypertension. The conventional analytical techniques to estimate serum creatinine are limited by its non-specificity, prevalent in the optical Jaffe assay, and the high costs, involved in the enzymatic assays. This has led to intensive research efforts in developing an accurate, robust, and sensitive sensor for estimating the concentration of creatinine in serum with cost-effective solutions. This underlies the focus of the thesis as it primarily discusses various approaches and challenges faced in developing the intended sensor. The primary challenge in estimation of serum creatinine is posed by its reduced concentration in the complex matrix of blood, which is constituted by varied proteins, whole cells, immunoglobulins, ions, and other metabolites. The electro-inactivity of creatinine further complicates the measurement by an electrochemical route. This necessitates selection of a redox probe that has an inherently high selectivity for creatinine to address both issues. In this thesis, we have explored non-enzymatic and enzymatic approaches for its detection. One of the non-enzymatic approaches, involves utilization of a transition metal – iron that has an affinity for creatinine. The other non-enzymatic approach involves electrochemical estimation of creatinine by picric acid that is already utilized in the optical Jaffe reaction. Both the approaches provide reliable estimation of creatinine in saline and prove the feasibility of estimation of the reduced concentrations of serum creatinine by non-enzymatic techniques. The enzymatic approach involves one-step hydrolysis of creatinine by creatinine deiminase. The resulting N-methylhydantoin is quantified by a highly selective transition metal-based redox probe – cobalt. This is a novel route for creatinine estimation that has provided reliable quantification in serum and even, whole blood. The enzymatic approach assures a higher sensitivity and specificity of detection in real samples. Another major issue that limits the clinical use of electrochemical biosensors for the analytes present in lower concentrations, is electrode fouling caused by the non-electroactive components of the blood. We have investigated different strategies to minimize this fouling by serum proteins – albumin. The strategies differ based on the interaction of the redox probe with albumin and are classified into albumin-reactive and albumin-non-reactive systems. Dilution with appropriate electrode surface modification has proven effective for albuminreactive systems. A unique size and charge filter composite has been devised for albumin-non-reactive systems. This filter composite can be tuned and further optimized with any disposable electrode platform, based on the required extent and nature of filtration. It also serves as a viable pre-treatment or activation layer for the lower cost ultramicroelectrodes. Decentralized testing necessitates minimal user involvement and no technical pre-requisites for the measurement. Hence, we have proposed a sequential drop technique of the sensing chemistry constituting cobalt ions on disposable screenprinted electrodes as a decentralized testing solution with minimal user involvement. We have also explored electrodeposition of cobalt ions as an alternate platform that further minimizes the user involvement by confinement of the sensing chemistry on the electrode surface. Thus, this thesis provides a comprehensive exploration of non-enzymatic and enzymatic techniques for quantification of serum creatinine. The device based on enzymatic technique has demonstrated success in estimation of creatinine from whole blood samples of patients with no sample pre-processing, a reduced turnaround time and high accuracy over a wide dynamic range and has laid the foundation for the intended point-of-care device.