Rapid and Label-Free Detection of Interferon Gamma via an Electrochemical Aptasensor Comprising a Ternary Surface Monolayer on a Gold Interdigitated Electrode Array

Abstract

A label-free electrochemical impedance spectroscopy (EIS) aptasensor for rapid detection (<35>min) of interferon-gamma (IFN-γ) was fabricated by immobilizing a RNA aptamer capture probe (ACP), selective to IFN-γ, on a gold interdigitated electrode array (Au IDE). The ACP was modified with a thiol group at the 5′ terminal end and subsequently co-immobilized with 1,6-hexanedithiol (HDT) and 6-mercapto-1-hexanolphosphate (MCH) to the gold surface through thiol-gold interactions. This ACP/ HDT-MCH ternary surface monolayer facilitates efficient hybridization with IFN-γ and displays high resistance to nonspecific adsorption of nontarget proteins [i.e., fetal bovine serum (FBS) and bovine serum albumin (BSA)]. The Au IDE functionalized with ACP/HDT-MCH was able to measure IFN-γ in actual FBS solution with a linear sensing range from 22.22 pM to 0.11 nM (1-5 ng/mL) and a detection limit of 11.56 pM. The ability to rapidly sense IFN-γ within this sensing range makes the developed electrochemical platform conducive toward in-field disease detection of a variety of diseases including paratuberculosis (i.e., Johne's Disease). Furthermore, experimental results were numerically validated with an equivalent circuit model that elucidated the effects of the sensing process and the influence of the immobilized ternary monolayer on signal output. This is the first time that ternary surface monolayers have been used to selectively capture/detect IFN-γ on Au IDEs. ABSTRACT: A label-free electrochemical impedance spectroscopy (EIS) aptasensor for rapid detection (<35 min) of interferon-gamma (IFN-γ) was fabricated by immobilizing a RNA aptamer capture probe (ACP), selective to IFN-γ, on a gold interdigitated electrode array (Au IDE). The ACP was modified with a thiol group at the 5′ terminal end and subsequently coimmobilized with 1,6-hexanedithiol (HDT) and 6-mercapto-1-hexanolphosphate (MCH) to the gold surface through thiol−gold interactions. This ACP/HDT-MCH ternary surface monolayer facilitates efficient hybridization with IFN-γ and displays high resistance to nonspecific adsorption of nontarget proteins [i.e., fetal bovine serum (FBS) and bovine serum albumin (BSA)]. The Au IDE functionalized with ACP/HDT-MCH was able to measure IFN-γ in actual FBS solution with a linear sensing range from 22.22 pM to 0.11 nM (1−5 ng/mL) and a detection limit of 11.56 pM. The ability to rapidly sense IFN-γ within this sensing range makes the developed electrochemical platform conducive toward in-field disease detection of a variety of diseases including paratuberculosis (i.e., Johne's Disease). Furthermore, experimental results were numerically validated with an equivalent circuit model that elucidated the effects of the sensing process and the influence of the immobilized ternary monolayer on signal output. This is the first time that ternary surface monolayers have been used to selectively capture/detect IFN-γ on Au IDEs.

Post hoc support vector machine learning for impedimetric biosensors based on weak protein–ligand interactions

“…A Randles-Ershler equivalent circuit (Chi 2 =1087 ± 212) was used to extract R s , R ct , Z w , C dl for each experiment (see supplemental Table S1 for details). Similar to other manuscripts in the literature 51 , R ct was used as the most accurate parameter for characterizing protein-biomolecule interactions (Fig 2b). For comparison, addition of BSA or buffer did not result in any significant change in impedance due to non-specific binding (see supplemental Figure S2).…”

A highly sensitive electrochemical IFN-γ aptasensor based on a hierarchical graphene/AuNPs electrode interface with a dual enzyme-assisted amplification strategy

“…Correspondingly, a linear regression equation of I peak ¼ 14.40 + 3.36 log C IFN-g (R 2 ¼ 0.995) with a low detection limit of 2 pM (S/N ¼ 3) was achieved. Although more analytical time was required comparing with some other reported IFN-g aptasensors (shown in Table S2 †), 17,18,[55][56][57][58][59] the performance of prepared aptasensor was comparable or better than most of reported biosensors. Besides, the proposed aptasensor could not be regenerated due to the involved target recycling strategy assisted with exonuclease.…”

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

“…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 …”

“…Wang's research group was pioneer in the development of ternary DNA SAM‐interfaces prepared by adding, instead of a short monothiol, a short dithiol as co‐adsorbent/backfiller to the common binary SHCP+MCH SAM ,,,,. These ternary SAMs were prepared by co‐immobilization of a short cyclic or linear,, dithiol with the SHCP, followed by chemisorption of MCH in a subsequent step, and provided large signal‐to‐blank ratios. This behavior was attributed to an efficient passivation of the free gold surface, probably related to a lying flat positioning of the dithiol covering cavities that are strong unspecific adsorption points, without sacrificing the electron transfer through the sensing layer.…”

“…In addition, the MCH was incorporated just 15 minutes before the control measurement and, therefore, the risk of DNA displacement was much lower. 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 …”