Cargando…

Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis

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 rat...

Descripción completa

Detalles Bibliográficos
Autores principales: Corrêa, Ricardo A. M. S., da Cruz, Filipe S., Santos, Cátia C., Pimenta, Thiago C., Franco, Diego L., Ferreira, Lucas F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165072/
https://www.ncbi.nlm.nih.gov/pubmed/30208638
http://dx.doi.org/10.3390/bios8030084
_version_ 1783359750587547648
author Corrêa, Ricardo A. M. S.
da Cruz, Filipe S.
Santos, Cátia C.
Pimenta, Thiago C.
Franco, Diego L.
Ferreira, Lucas F.
author_facet Corrêa, Ricardo A. M. S.
da Cruz, Filipe S.
Santos, Cátia C.
Pimenta, Thiago C.
Franco, Diego L.
Ferreira, Lucas F.
author_sort Corrêa, Ricardo A. M. S.
collection PubMed
description 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.
format Online
Article
Text
id pubmed-6165072
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-61650722018-10-10 Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis Corrêa, Ricardo A. M. S. da Cruz, Filipe S. Santos, Cátia C. Pimenta, Thiago C. Franco, Diego L. Ferreira, Lucas F. Biosensors (Basel) Article 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. MDPI 2018-09-11 /pmc/articles/PMC6165072/ /pubmed/30208638 http://dx.doi.org/10.3390/bios8030084 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Corrêa, Ricardo A. M. S.
da Cruz, Filipe S.
Santos, Cátia C.
Pimenta, Thiago C.
Franco, Diego L.
Ferreira, Lucas F.
Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title_full Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title_fullStr Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title_full_unstemmed Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title_short Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis
title_sort optimization and application of electrochemical transducer for detection of specific oligonucleotide sequence for mycobacterium tuberculosis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6165072/
https://www.ncbi.nlm.nih.gov/pubmed/30208638
http://dx.doi.org/10.3390/bios8030084
work_keys_str_mv AT correaricardoams optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis
AT dacruzfilipes optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis
AT santoscatiac optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis
AT pimentathiagoc optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis
AT francodiegol optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis
AT ferreiralucasf optimizationandapplicationofelectrochemicaltransducerfordetectionofspecificoligonucleotidesequenceformycobacteriumtuberculosis