Tetrasiklin Tayinine Yönelik Aptamer Bağlı Peroksidaz Benzeri Mikropartiküllerin Geliştirilmesi

Abstract

Sensitive and selective methods are needed for the determination of antibiotic residues accumulated in food and water sources to protect human safety and health. Traditional methods used for determination of tetracycline residues, which are widely used, have disadvantages such as being time consuming, requiring professional working skills, and using expensive devices for determination. A biosensor including aptamer and a magnetic microparticle with peroxidase-like activity was developed for tetracycline detection in aqueous solutions. In this study, magnetic microparticle with peroxidase-like activity was developed as an alternative to tetracycline determination methods currently used. Within this scope, (3-glycidyloxypropyl)trimethoxysilane (GLYMO)-linked MagSiO2 microparticles and (3-aminopropyl)triethoxysilane (APTES)-linked MagSiO2 microparticles were synthesized. For the synthesis of GLYMO-linked MagSiO2 microparticles, magnetic silica microparticles MagSiO2, toluene and GLYMO were reacted under reflux at 110°C for t=8 hours. Microparticles were washed with ethanol, ddH2O and sodium carbonate (Na2CO3), respectively. After that, microparticles were reacted with ethylenediamine and Na2CO3 at 60°C for t=24 h, washed with phosphate buffer (PB) and ddH2O. After determination of particle density by gravimetric analysis, microparticles were washed with PB again. For the synthesis of APTES-linked MagSiO2 microparticles, (APTES), magnetic silica microparticles (MagSiO2), toluene and tris-ethylamine were used in a similar synthesis protocol. O-phenylenediamine (OPD) was used as substrate in the peroxidase-like activity measurements. Measurements showed that the absorbance value of GLYMO-linked MagSiO2 microparticles was greater than APTES-linked MagSiO2 microparticles. GLYMO-linked MagSiO2 microparticles and glutaraldehyde crosslinker were reacted in the dark, at room temperature for 18-24 hours. After the reaction, microparticles were washed with PB. Aptamers were denatured at 95°C for t=5 min, followed by a cooling step on ice for t=10 min. After that, aptamers and microparticles were reacted at room temperature for t=2 h. After the mixture of aptamer and microparticles were washed with PB, the mixture was reacted with tetracycline solutions (100-200-500-1000-2000 ng/μl) at room temperature for t=30 min and than washed with PB. Thereafter, the mixture was reacted in the dark with OPD at room temperature for t=45-75 min. The effect of aptamer concentrations and aptamer folding within buffer solutions having different ions and ion concentrations on tetracycline determination was also studied. Amount of H2O2 solution, medium buffer solution and it’s pH value were optimized for the OPD solution. Absorbance values were measured at 450 nm in a plate reader, and 10 mM OPD solution prepared with phosphate citrate buffer solution with pH=7 had greater ΔA than other OPD solutions. The optimum reaction time was obtained for t=60 min. To stop the reaction of OPD, 3 M HCL was used. According to absorbance values measured at 492 nm, as the amount of HCl was increased, the absorbance values increased, but ΔA values decreased.

After all the optimizations, tetracycline concentrations were scanned and a limit of detection LOD=100 ng/μl was obtained, while the maximum residue limits of tetracycline allowed in milk are 200 ng/μl in the European Union. Overall, an easy, inexpensive, aptamer-linked, enzyme-like magnetic particle based biosensor has been developed towards tetracycline detection.