Optimization and application of electrochemical transducer for detection of specific oligonucleotide sequence for mycobacterium tuberculosis

Abstract

In this study, the electropolymerization of 4-hydroxyphenylacetic acid (4-HPA) over graphite electrodes (GE) was optimized, aiming its application as a functionalized electrochemical platform for oligonucleotides immobilization. It was investigated for the number of potential cycles and the scan rate influence on the monomer electropolymerization by using cyclic voltammetry technique. It was observed that the polymeric film showed a redox response in the region of +0.53/+0.38 V and the increase in the number of cycles produces more electroactive platforms because of the better electrode coverage. On the other hand, the decrease of scan rate produces more electroactive platforms because of the occurrence of more organized coupling. Scanning electron microscopy (SEM) images showed that the number of potential cycles influences the coverage and morphology of the electrodeposited polymeric film. However, the images also showed that at different scan rates a more organized material was produced. The influence of these optimized polymerization parameters was evaluated both in the immobilization of specific oligonucleotides and in the detection of hybridization with complementary target. Poly(4-HPA)/GE platform has shown efficient and sensitive for oligonucleotides immobilization, as well as for a hybridization event with the complementary oligonucleotide in all investigated cases. The electrode was modified with 100 cycles at 75 mV/s presented the best responses in function of the amplitude at the monitored peak current values for the Methylene Blue and Ethidium Bromide intercalators. The construction of the genosensor to detect a specific oligonucleotide sequence for the Mycobacterium tuberculosis bacillus confirmed the results regarding the poly(4-HPA)/GE platform efficiency since it showed excellent sensitivity. The limit of detection and the limit of quantification was found to be 0.56 (±0.05) μM and 8.6 (±0.7) μM, respectively operating with very low solution volumes (15 μL of probe and 10 μL target). The biosensor development was possible with optimization of the probe adsorption parameters and target hybridization, which led to an improvement in the decrease of the Methylene Blue (MB) reduction signal from 14% to 34%. In addition, interference studies showed that the genosensor has satisfactory selectivity since the hybridization with a non-specific probe resulted in a signal decrease (46% lower) when compared to the specific target.