Otimização de um biossensor “label-free” através da espectroscopia de impedância eletroquímica para detecção de DNA

The clear need forlow-cost portable biosensor continues to drive significant research fromdifferent fields. DNA biosensors are demand in many areas including diagnostic, genomic and forensics [1]. Withal, electrochemical biosensor hasbeen extensivelyused due to its capacity for rapid and accurate detection of a wide variety of target molecules or biomarkers.Therefore, electrochemical DNA biosensor haspotential to fulfill thisdemand. However, the development of this platformrequires first anoptimum surface densityof probes to achieve lower limits of detection.In this study we used a mixedself-assembled monolayer (SAM) formed by simultaneous co-immobilization of thiol modified DNA probesand mercaptohexanol(MCH)onto gold electrodes.The probe surface density was optimizedto obtain the maximum change in charge transfer resistance with hybridization.For this,the electrodes were immersed in a buffer with hexaammineruthenium (III) chloride ([Ru(NH3)6]+3).The DNA surface densitywas calculated from the number of cationic redox molecules electrostatically associated with the anionic DNA backboneusing chronocoulometry [2]. The amount of [Ru(NH3)6]+3was determined integrating the current as a function of time,given by the integrated Cottrell equation and presented a linear relationship between thiol molar ratio andprobe densityfrom 2 to 5 x1012/cm2. The effect of hybridization was determined using electrochemical impedance spectroscopy (EIS) withnegatively charged ferri/ferrocyanide redox couple in solution. The immobilized DNA has negative charges due to phosphate groups and, consequently, anelectrostatic repulsion withnegatively chargedredox couple, resultingin a modulationof the charge transfer resistance(Rct). After probe surface density optimization, the maximum shift of Rct upon 1µM complementary sequence was obtained with around 30% probe fraction immobilized on surface. Thiselectrochemical platform developed was able to distinguemismatched target sequences and can be expanded to other detections.