Determination of Integrity, Stability and Density of the DNA Layers Immobilised at Glassy Carbon and Gold Electrodes Using Ferrocyanide

Banasiak, Anna; Colleran, John

doi:10.1002/elan.202060077

“…However, the small size limits their application in the field of electrochemical biosensing. Because the sensitivity of biosensors largely depends on the amount and density of the target or electrochemically active substances that can be effectively captured or reacted at the electrode surface [142,143]. Therefore, exploiting the signal amplification technology by improving the electrochemically active area or recycling the electroactive production is an urgent issue to improve the sensitivity of the POCT based on the microfabricated electrochemical device.…”

Section: Signal Amplification Technologymentioning

confidence: 99%

Electrochemical Biosensors Based on Micro‐fabricated Devices for Point‐of‐Care Testing: A Review

Zhang

¹

,

Zhou

²

2021

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Point-of-care testing (POCT) is becoming a hot research topic that allows rapid, on-site, and non-professional measurements outside the central laboratory. The micro-fabricated devices prepared by various micromachining technologies have shown the advantages of low reagent consumption, high-throughput samples, and wearability. This review presents the recent progress of electrochemical biosensors based on various micro-fabri-cated devices for POCT and the corresponding electrochemical techniques. Signal amplification strategies based on enzyme and nanotechnology are also illustrated for the more sensitive POCT applications of these micro-fabricated devices. Consequently, the trends and challenges of electrochemical biosensors based on micro-fabricated devices in POCT diagnosis are discussed.

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“…210,211 The main advantages of the ferri/ferro couple are the following: it exhibits reversible heterogeneous kinetics, it has stable oxidized and reduced forms, it is soluble in aqueous solution, and, in aptasensors field, it doesn't react or interact with DNA. 209,212,213 In a standard e-chem aptasensor system using the ferri/ferro couple, the aptamer is attached covalently to the gold electrode via an Au-S bond. Lin et al 214 developed a label-free e-chem aptasensor for 17β-estradiol.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

Sester¹

0

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Aptamers are synthetic nucleic acid single-stranded oligonucleotides that bind to a wide range of ligands, including cells, proteins, DNA strands, metal ions, and small molecules, with high specificity and affinity. Aptamers have also proven to be highly stable, readily adaptable to chemical modifications, and exhibit reversible binding. As a result, aptamer-based biosensors (aptasensors) are promising replacements for antibody-based biosensors in many applications, particularly for small molecule ligands. This thesis explores an aptamer that binds the drug methamphetamine, and its prospects when incorporated in an electrochemical (e-chem) signal transduction platform. Specifically, we examine the range of interactions between the aptamer and ligand, and with electrodes, and identify a number of challenges in generating robust e-chem aptasensors. Due to their size and limited number of functional groups, further understanding of the aptamer-small molecule ligand interactions is required for the design of future aptasensors – particularly the thermodynamics and structural information about the aptamer-ligand interaction. In fact, detecting small molecules with aptasensors can become challenging because target addition may induce little structural change, and therefore numerous nonspecific interactions may emerge as transduced signals from the biosensor. In this thesis, the combination of spectroscopic and calorimetric analytical techniques reveals a conformational selection binding model, in which binding is entropically driven, and the meth binds via hydrophobic and electrostatic interactions and only induces a modest structural change. This first-of-its-kind study is important for the selection and the design of the aptasensor transduction system. Electrochemical (e-chem) aptasensors offer high inherent sensitivity and practicality as a signal transduction platform. Indeed, different e-chem aptasensor formats have been published before, including labelled and label-free sensors. In screening the viability of three commonly used methods – including labelled and label-free, as well voltammetric and impedance-based methods – we find that each of them suffers from instability of the aptamer-functionalised electrode. This instability compromises our ability to resolve real signals and prompted us to develop ways to understand – and suppress – this baseline drift. The functionalization of the electrode is the critical step in terms of self-assembled monolayer (SAM) stability and SAM aptamer density. Consequently, different protocols of SAMformation were explored and evaluated with respect to stability. We find that instability arises from the uncontrolled arrangement of thiolated aptamers on gold electrodes (including aptamers lying down on the electrode), which is in turn affected by the density of aptamers that can be coupled to the surface. As a consequence, a new protocol is developed using disulfide aptamer pairs to increase the density of correctly tethered aptamers, and generate a stable SAM. Because of the high sensitivity of electrochemical platforms, numerous spurious electrochemical signals may be produced, and controlled for in order to confirm a positive ligand-binding signal. The specificity of the aptamer-target interaction can be checked by testing the response with an interferent molecule, or by substituting the aptamer with a non-binding nucleotide sequence. In this work, these control experiments reveal that target and interferent molecules interact directly (and in different ways) with the bare gold surface, as well as perturbing signals from the aptamer in ways that cannot be linked to a specific aptamer-ligand complex formation. Ultimately, these spurious signals compromise our ability to confirm a real binding signal. The results from this work provide the first clear picture of how an aptamer binds to its small molecule target – which we find is entropically driven, and with only minor structural change induced in the aptamer stem. In addition, the label-free EIS measurements on aptamer SAM electrodes reveal the nature of instabilities, and reveal spurious signals that cannot be sufficiently suppressed at this stage. This knowledge highlights the difficulty in fabricating e-chem aptasensors, and will assist in overcoming challenges faced during research and commercialization of aptasensors area, as well as contributing new insights into troubleshooting, data acquisition, and data validation.

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Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

Sester¹

2021

Preprint

0

View full text Add to dashboard Cite

Aptamers are synthetic nucleic acid single-stranded oligonucleotides that bind to a wide range of ligands, including cells, proteins, DNA strands, metal ions, and small molecules, with high specificity and affinity. Aptamers have also proven to be highly stable, readily adaptable to chemical modifications, and exhibit reversible binding. As a result, aptamer-based biosensors (aptasensors) are promising replacements for antibody-based biosensors in many applications, particularly for small molecule ligands. This thesis explores an aptamer that binds the drug methamphetamine, and its prospects when incorporated in an electrochemical (e-chem) signal transduction platform. Specifically, we examine the range of interactions between the aptamer and ligand, and with electrodes, and identify a number of challenges in generating robust e-chem aptasensors. Due to their size and limited number of functional groups, further understanding of the aptamer-small molecule ligand interactions is required for the design of future aptasensors – particularly the thermodynamics and structural information about the aptamer-ligand interaction. In fact, detecting small molecules with aptasensors can become challenging because target addition may induce little structural change, and therefore numerous nonspecific interactions may emerge as transduced signals from the biosensor. In this thesis, the combination of spectroscopic and calorimetric analytical techniques reveals a conformational selection binding model, in which binding is entropically driven, and the meth binds via hydrophobic and electrostatic interactions and only induces a modest structural change. This first-of-its-kind study is important for the selection and the design of the aptasensor transduction system. Electrochemical (e-chem) aptasensors offer high inherent sensitivity and practicality as a signal transduction platform. Indeed, different e-chem aptasensor formats have been published before, including labelled and label-free sensors. In screening the viability of three commonly used methods – including labelled and label-free, as well voltammetric and impedance-based methods – we find that each of them suffers from instability of the aptamer-functionalised electrode. This instability compromises our ability to resolve real signals and prompted us to develop ways to understand – and suppress – this baseline drift. The functionalization of the electrode is the critical step in terms of self-assembled monolayer (SAM) stability and SAM aptamer density. Consequently, different protocols of SAMformation were explored and evaluated with respect to stability. We find that instability arises from the uncontrolled arrangement of thiolated aptamers on gold electrodes (including aptamers lying down on the electrode), which is in turn affected by the density of aptamers that can be coupled to the surface. As a consequence, a new protocol is developed using disulfide aptamer pairs to increase the density of correctly tethered aptamers, and generate a stable SAM. Because of the high sensitivity of electrochemical platforms, numerous spurious electrochemical signals may be produced, and controlled for in order to confirm a positive ligand-binding signal. The specificity of the aptamer-target interaction can be checked by testing the response with an interferent molecule, or by substituting the aptamer with a non-binding nucleotide sequence. In this work, these control experiments reveal that target and interferent molecules interact directly (and in different ways) with the bare gold surface, as well as perturbing signals from the aptamer in ways that cannot be linked to a specific aptamer-ligand complex formation. Ultimately, these spurious signals compromise our ability to confirm a real binding signal. The results from this work provide the first clear picture of how an aptamer binds to its small molecule target – which we find is entropically driven, and with only minor structural change induced in the aptamer stem. In addition, the label-free EIS measurements on aptamer SAM electrodes reveal the nature of instabilities, and reveal spurious signals that cannot be sufficiently suppressed at this stage. This knowledge highlights the difficulty in fabricating e-chem aptasensors, and will assist in overcoming challenges faced during research and commercialization of aptasensors area, as well as contributing new insights into troubleshooting, data acquisition, and data validation.

show abstract

Determination of Integrity, Stability and Density of the DNA Layers Immobilised at Glassy Carbon and Gold Electrodes Using Ferrocyanide

Cited by 3 publications

References 75 publications

Electrochemical Biosensors Based on Micro‐fabricated Devices for Point‐of‐Care Testing: A Review

Electrochemical Biosensors Based on Micro‐fabricated Devices for Point‐of‐Care Testing: A Review

Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

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