Electrochemical platforms for solid-phase isothermal amplification and detection of bacterial genome Raquel Sánchez-Salcedo, R. Miranda-Castro, N. de-los-Santos-Álvarez, M. J. Lobo-Castañón Universidad de Oviedo Departamento de Química Física y Analítica 1st International Electronic Conference on Biosensors
Introduction Salmonella as many bacterial pathogens constitutes a mayor cause of foodborne disease 1st International Electronic Conference on Biosensors
Introduction Monitoring and control of this human pathogenic bacterium in foodstuffs and biological fluids are necessary in order to prevent and diagnose the disease Denaturation (95 °C) Highly sensitive Reliable PCR Sophisticated equipment Difficult to miniaturize Time-consuming Elongation Annealing (72 °C) (55-65 °C) 1st International Electronic Conference on Biosensors
Introduction Alternative to PCR Isothermal nucleic acid amplifications • Constant temperature • Short times • Simple equipment Helicase-dependent amplification (HDA) Recombinase polymerase amplification (RPA) 1st International Electronic Conference on Biosensors
Introduction On-surface isothermal amplifications Overcome challenges Point-of-need devices Electrochemical platform + Isothermal amplification • Inexpensive • Simple • Portable • Compatible with microfluidic technologies 1st International Electronic Conference on Biosensors
Objectives Comparison of two electrochemical platforms to detect Salmonella genome Solid-phase RPA onto Au surface Solid-phase HDA onto ITO surface POD 1-Naphthol DPV Chronoamperometry Indium-tin oxide (ITO) surfaces Gold surfaces 6-FAM-Forward primer antiFITC-POD antiFITC-AP HS-T x -Reverse primer 1st International Electronic Conference on Biosensors
Results & discussion Sensing phase construction for HDA onto ITO NH 2 Electrochemical set-up Si OH OH OH O O O H 2 O 2 /NH 4 OH/H 2 O 1 % APTES (RT, overnight) (RT, 1 h) ITO surface S O 1. N (RT, 1 h, in darkness) 2. HS HS-T 10 -RP, (RT, 2 h) Si O O O 1st International Electronic Conference on Biosensors
Results & discussion Sensing phase construction for RPA onto gold Electrochemical set-up Reference electrode: Ag/AgCl/KCl (3 M) electrode isolated from the test solution by a KNO3 (3 M) salt bridge inside a syringe HS HS-T 15 -RP S (4 °C, overnight) Au surface HS NH 2 NH 2 NH 2 (RT, 1h) S S Sensing platform Gold counter electrode Gold working electrode 1st International Electronic Conference on Biosensors
Results & discussion Helicase-dependent amplification (HDA) in solution (SSBP) Single-strand binding protein * G. A. Obande and K. K. B. Singh, Infect. Drug Resist. , 13 (2020) 455 – 483. 1st International Electronic Conference on Biosensors
Results & discussion HDA onto ITO surfaces A) First stage: B) Second stage: Amplification starts in Amplification takes place solution, giving rise to on surface boosted by an 86 bp product the immobilized primer *Barreda-García, S.; Miranda-Castro, R.; de-los-Santos-Álvarez, N.; Miranda-Ordieres, A.J.; Lobo-Castañón, M.J., Chem. Commun. 2017 , 53 , 9721 – 9724, 1st International Electronic Conference on Biosensors
Results & discussion Recombinase polymerase amplification (RPA) in solution 1. Formation of recombinase-primer complex 2. Homologous sequence recognition 3. Stabilization by single-strand DNA binding proteins (SSBs) 4. DNA polymerase elongation *J. Li and J. Macdonald Biosensors and Bioelectronics, 69 (2015) 196-211 1st International Electronic Conference on Biosensors
Results & discussion On-gold RPA 1. Hybridization between genome and the attached primer 2. Elongation of the primer 3. Label-free amplicons 4. Second surface amplification 5. Incorporation of the tag 6. Labeled inmobilized amplicon HS-T 15 -Reverse primer Target DNA 6-FAM-Forward primer *Sánchez-Salcedo, R.; Miranda-Castro, R.; de-los-Santos-Álvarez, N.; Lobo-Castañón, M.J., ChemElectroChem 2019 , 6 , 793 – 800. 1st International Electronic Conference on Biosensors
Results & discussion Evaluation of the analytical performance of both platforms Features related to the surface 1-Naphthol HDA RPA Temperature (°C) 65 37 Surface ITO Gold Enzyme conjugate AntiFab-AP AntiFab-POD Enzyme substrate 1-naphthyl phosphate TMB Detection technique DPV Chronoamperometry Storage stability 9 months 1 month Chronoamperometry Gold 1st International Electronic Conference on Biosensors
Results & discussion Evaluation of the analytical performance of both platforms Features related to the amplification 90 min HDA RPA 65 °C Temperature (°C) 65 37 37 °C Available format Kit Kit 30 min Time (min) 90 30 10 5 LOD (genomes) 10 Reproducibility (%) 20 30 1st International Electronic Conference on Biosensors
Conclusions 1. The devices compared in this work successfully integrate isothermal amplification in electrochemical platforms 2. Achieving efficient detection of small amounts of the Salmonella genome without thermal cycling while using simple equipment is accomplished in both cases. 3. The results of this study may be of general utility in the design of sensors for detecting other bacteria . 1st International Electronic Conference on Biosensors
Acknowledgement Electroanalysis group MSc. Raquel Sánchez-Salcedo MSc. Ana Díaz-Fernández MSc. Ramón Lorenzo-Gómez MSc. Clara Abardía-Serrano Dr. Rebeca Miranda-Castro Dr. Noemí de-los-Santos-Álvarez Prof. María J. Lobo-Castañón RTI-2018-095756-B-I00 IDI/2018/0000217 1st International Electronic Conference on Biosensors
Electrochemical platforms for solid-phase isothermal amplification and detection of bacterial genome Raquel Sánchez-Salcedo, R. Miranda-Castro, N. de-los-Santos-Álvarez, M. J. Lobo-Castañón Universidad de Oviedo Departamento de Química Física y Analítica 1st International Electronic Conference on Biosensors
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