Key Factors of Ternary Monolayers to Improve DNA Sensors Performance

Abstract

An important factor affecting the performance of electrochemical genosensors is the immobilization strategy used to anchor the capture probes (CP) on the electrode surface. One of the most common immobilization systems involves the formation of self‐assembled monolayers of thiolated probes on gold surfaces, mainly due to its simplicity and effectiveness. Recent studies have shown that in two‐component thiolated DNA and mercaptohexanol (MCH) monolayers, there is incomplete backfilling that leads to defective surface blockage, producing unspecific background signal. In order to solve these problems, ternary monolayers have been proposed, in which besides MCH as primary diluent, a second diluent is added to enhance the hybridization efficiency and anti‐fouling properties of the surface. Herein, we report a thorough evaluation of different surface chemistries using two different sensors, one for the detection of a peanut allergen encoding sequence and the other one targeting a gluten‐encoding sequence. We assessed the relationship between CP length and the chemical characteristics of the diluents. Factors such as concentration, nature of the functional groups and chain length of the second diluent were evaluated. We found that length of diluents affects primarily the hybridization efficiency, while the type of functional groups, e.g. hydroxyl, carboxyl or a second thiol group, influences the degree of surface coverage. Results diverged between the two sensors evaluated.

Tailoring Sensitivity in Electrochemical Nucleic Acid Hybridization Biosensing: Role of Surface Chemistry and Labeling Strategies

“…Gold‐alkanethiol chemistry provides highly ordered and pinhole free surfaces allowing the construction of sensing platforms with increased flexibility and stability . Thus, self‐assembled monolayers (SAMs) is a widely used immobilization strategy for thiol‐tethered DNA probes, ssDNA‐HS, in the design of DNA biosensors . Within this context, it is well known that electrodes modified only with capture probe strands provide little or no discrimination after hybridization with the target nucleic acid sequence due to the inappropriate lying down conformation of the probe molecules.…”

“…The use of ternary mixed monolayers comprising two different thiols as diluents/spacers together with an appropriate SHCP self‐assembled on gold electrodes provides interfaces with extremely attractive characteristics for electrochemical biosensing of target nucleic acids hybridization,,,, and aptasensing …”

“…Due to the MCH‐ssDNA hydrophobic interactions against MPA‐ssDNA hydrophilic interactions, MPA was shown to be more efficient than MCH in reducing unspecific adsorptions and placing the DNA strands perpendicularly to the electrode surface while retaining intact the molecular environment surrounding HS‐ssDNA . Authors demonstrated that the combination of MCH and MPA generated hydrogen bonds (each MPA molecule bonds with two MCH) thus forming a pseudo‐layer of hydrogen bonds preventing the access of the redox species ([Fe(CN) 6 ] 3− ) to the electrode surface and decreasing the unspecific adsorptions (Figure ) ,…”

“…These monolayers have been assembled onto photolithographically prepared 16‐Au electrode arrays,,,, Au interdigitated electrode array and commercial screen‐printed Au electrodes . The as prepared DNA biosensors exhibited great performance for the electrochemical detection of relevant nucleic acids including bacterial rRNA,,,,,, peanut allergen and gluten‐encoding DNA sequence, precursor rRNAs (pre‐rRNAs) and proteins such IFN‐γ …”

“…Exhaustive optimizations with ternary mixed monolayers showed that factors such as the fabrication protocol, concentration of the components, nature of the functional groups and chain length of the second diluent are key parameters in the resulting performance of the biosensors affecting primarily the hybridization efficiency (length of diluents), degree of surface coverage (type of functional groups, e. g. hydroxyl, carboxyl or a second thiol group) and non‐specific adsorptions ,. Recently, it has been reported that the stability of these monolayers (including also the binary ones) can be improved by keeping them in the presence of stabilizing agents such as BSA in combination with saccharide molecules as trehalose and glucose).…”