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Development of amperometric immunosensor for diagnosis of albuminuria based on screen-printed electrodes

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MIKUŠOVÁ Zuzana PASTUCHA Matěj FARKA Zdeněk POLÁCHOVÁ Veronika SKLÁDAL Petr

Druh Článek ve sborníku
Konference XVIII. Workshop of Biophysical Chemists and Electrochemists
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
WWW http://www.sci.muni.cz/~labifel/files/soubory/sbornik_2018.pdf#page=33
Klíčová slova Biosensor; Amperometry; Sandwich assay; Human serum albumin; Albuminuria
Popis Human serum albumin (HSA) is a plasma protein which plays an important role in transporting ligands to specific targets. Increased presence of HSA in urine (microalbuminuria) is considered as an indication of diabetic nephropathy or glomerulonephritis. The amount of HSA in 24-h urine specimens typical for microalbuminuria ranges from 30 to 300 mg. Nowadays, there are various immunoassays for detection of HSA such as enzyme linked immunosorbent assay (ELISA). These methods provide high sensitivity; however, they are time consuming, complicated and require expensive instrumentation. An appropriate solution to overcome these disadvantages is the use of electrochemical biosensors that offer operational simplicity, high sensitivity, short analysis times and miniaturization possibilities for point-of-care diagnosis. The electrochemical biosensors are typically based on screen-printed electrodes (SPEs) due to their low cost, portability and mass production capabilities. We have developed an amperometric immunosensor for rapid detection of HSA in urine. Anti-HSA antibody was immobilized to the surface of electrodes that allowed specific capture of HSA. The detection was based on sandwich immunoassay principle where the secondary antibody conjugated with horseradish peroxidase (HRP) was binding to surface captured immunocomplex Ab1-HSA. HRP as label provides oxidation of 3,3´,5,5´-tetramethylbenzidine (TMB) in presence of H2O2. Alternatively, we used Prussian blue catalytic nanoparticles as convenient enzyme replacement. Non-specific binding was evaluated by incubating the sensor with BSA instead of HSA. We compered SPEs based on gold and carbon (including carbon nanotubes) working electrodes. So far, the lowest concentration of detected HSA was 10 µg·mL-1. The actual limit of detection is useful for practical analysis and point-of-care diagnosis. This method has proved to be suitable for diagnostics of microalbuminuria. Optimization focused on better detection limit and to reduce, respectively eliminate non-specific binding is in progress.
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