Spectroelectrochemical Detection of Reduced Water-soluble Tetrazolium Salts Using Disposable Pencil Graphite Electrodes

Microalbuminuria is an important clinical biomarker of diabetes mellitus, chronic kidney disease (CKD), and diabetic KD. However, microalbuminuria detection in urine by immunoturbidimetric method, for early detection and follow-up, is a complicated and expensive procedure compared to the qualitative and semi-quantitative methods, such as the most commonly used dipstick urinalysis. Here, we developed an enzyme- and antibody-free approach using the redox reaction of water-soluble tetrazolium salts (WSTs), which were reduced by albumin and protein and finally produced the colored formazan. Colorimetry was used to spectrometrically detect blue formazan, a reduced WST, and the absorbance was measured at about 600 nm using a microplate reader. The calibration curve for the standard solutions showed that the assay is linear between 0 and 150 mg/L albumin. A simple, rapid, and low-cost fabrication method is proposed here for assembling an electrochemical detection system for formazan using disposable pencil graphite electrodes (PGEs), which are the mechanical pencil leads as working, reference, and counter electrodes. The distinctive voltammetric oxidation peak appeared at +1250 mV vs. PGE by linear sweep voltammetry using a potentiostat. The amperometric oxidation peak current of formazan was applied at +1250 mV vs. PGE by chronoamperometry which required only a few seconds. A correlation was obtained between the oxidation peak maximum current of formazan and concentrations of albumin.