Ready-To Diagnostic Kit Based on Electrochemical Nanobiosensor for Antibiotic Resistance Gene Determination

Abstract

Nowadays, antibiotic resistance, which occurs as a result of mutations in bacterial genes due to the frequent use of antibiotics, causes serious health problems and deaths. One of the resistance mechanisms that develop with the use of antibiotics is that bacteria begin to produce carbapenemase enzymes that cause multiple drug resistance in bacteria by hydrolyzing antibiotics. In this study, an alternative diagnostic kit method to traditional tests was developed for the determination of bacteria carrying relevant antibiotic resistance genes because these analyzes have multi-step sample preparation, long/complex determination schemes and are high cost. With the diagnostic kit containing a stable electrochemical nanobiosensor developed in this study, the presence of two different antibiotic resistance gene regions of the carbapenemase enzyme named OXA-48 and VIM, which are responsible for more than 50 % of deaths due to antibiotic resistance, was investigated. Carbon nanotubes (CNTs) has been modified to the pencil graphite electrode (PGE), then the synthetic capture probe DNAs are immobilized covalently to the surface containing nanomaterial. Later, the sensor surface, whose stability was ensured by surface blockage, thus gained "diagnostic kit feature" and was stored in the refrigerator (+4 degrees C) until analysis. Thus, the preparation of a ready-to-analyze diagnostic kit, which will provide detection without any preparation, has been completed. With the developed kit, it was possible to determine the resistance gene with 2.5 pmole/50 mu L of detection limit. In addition, analyzes with real samples could be performed even after 150 days (5 months) of kit preparation. This represents the first diagnostic kit system containing PGE and CNT for the detection of antibiotic resistance based on electrochemical alpha naphthol signal measurement in real samples with symmetrical and asymmetric structures (PCR). In the developed alternative test platform, the sample volume required for determination is 50 mu L and the analysis time with this portable open-use and throw-away diagnostic kit is 30 min.