Ternary Self-assembled Monolayer Research Articles

Aptasensor based on screen-printed electrode for breast cancer detection in undiluted human serum

Breast cancer remains one of the leading causes of women death. The development of more sensitive diagnostic tests, which could present a faster response, lower cost, and could promote early diagnosis would increase the chances of survival. This study reports the development and optimization of an electrochemical aptasensor for the detection of HER2 protein, a breast cancer biomarker. Two sensing platforms were developed on gold screen-printed electrodes. The first platform is composed of self-assembled monolayer (SAM) made from mixture of thiolated DNA aptamers specific for HER2 and 1-mercapto-6-hexanol (MCH), while the second one is a ternary SAM composed of the same aptamer and 1,6-hexanethiol (HDT). Both platforms were further passivated with MCH and blocked with bovine serum albumin. The biosensors were characterized using electrochemical impedance spectroscopy to detect the target protein from 1 pg/mL to 1 μg/mL in phosphate buffered saline, diluted and undiluted human serum through charge transfer resistance value. The ternary SAM architecture shows a reduction of non-specific attachment to the electrode surface due to the HDT antifouling properties. In addition, this platform exhibits 172 pg/mL as limit of detection and a sensitivity of 4.12% per decade for undiluted serum compared with SAM architecture with the 179 pg/mL and 4.32% per decade, respectively. Electrochemical aptasensors are highly promising for medical diagnostic and ternary layers could improve the limit of detection.

Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity

Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis–Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin cleavage. This indicates that a PEG-based peptide, employing methylene blue as redox reporter, and deposited on an electrode as a ternary SAM configuration, is a suitable platform to develop clinically-relevant and quantitative electrochemical peptide-based protease biosensing.

Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors.

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.

A reusable and label-free supersandwich biosensor for sensitive DNA detection by immobilizing target-triggered DNA concatamers on ternary self-assembled monolayer

A reusable and label-free supersandwich biosensor was constructed for sensitive DNA detection by immobilizing target-triggered DNA concatamers with redox-active intercalators on ternary self-assembled monolayer (TSAM). Interestingly, the target DNA (T-DNA) could hybridize with the inert dumbbell-shaped DNA (D-DNA) to form a duplex DNA containing two sticky termini. The duplex DNA was then hybridized with the capture DNA (C-DNA) and the DNA concatamer reaction proceeded. The ternary self-assembly method was chosen to obtain the low-density C-DNA without reciprocal winding on the gold electrode for high-efficient concatamers hybridization. Hexaammineruthenium chloride was herein used as an electrochemical probe and could intercalate into the groove of double-helix DNA via electrostatic effect. The resultant supersandwich biosensor showed a high sensitivity for the T-DNA detection and a linear dependence between the reduction peak currents and logarithm of T-DNA concentrations in the range of 100.0 fM–10.0nM with a relatively low detection limit of 30.0 fM. Moreover, the proposed biosensor exhibited favorable specificity and regenerability, and provided a new alternative to detect various target analytes by changing the sequence of capture probe and dumbbell probes, holding great potential for early diagnosis in gene-related diseases.

Impact of nanografting on the local structure of ternary self-assembled monolayers

Prior studies using single and binary adsorbates indicate that nanografting impacts the reaction pathways and local structure of self-assembled monolayers (SAMs). This work explores the influence of nanografting in the case of ternary SAMs. Using atomic force microscopy (AFM) as both a nanografting and imaging tool, the local structures of two ternary SAMs, SC14:SSC10CHO:SC2COOH and SC18:SSC10CHO:SC2COOH, formed under natural growth and nanografting were imaged and compared. The results indicate that nanografting impacts the degree of phase segregation and the domain height in ternary SAMs. In addition to the previously known effect of altering self-assembly pathways, this study reveals an additional impact for these ternary systems: By shaving over the previous trajectory (grafted region), nanografting could start exchange reactions and lateral movement of surface-bound thiols, which leads to new and somewhat unanticipated local structures.

Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform

Precursor rRNA (pre-rRNA) is an intermediate stage in the formation of mature rRNA and is a useful marker for cellular metabolism and growth rate. We developed an electrochemical sensor assay for Escherichia coli pre-rRNA involving hybridization of capture and detector probes with tail sections that are spliced away during rRNA maturation. A ternary self-assembled monolayer (SAM) prepared on gold electrode surfaces by coassembly of thiolated capture probes with hexanedithiol and posttreatment with 6-mercapto-1-hexanol minimized the background signal and maximized the signal-to-noise ratio. Inclusion of internal calibration controls allowed accurate estimation of the pre-rRNA copy number per cell. As expected, the ratio of pre-rRNA to mature rRNA was low during stationary phase and high during log phase. Pre-rRNA levels were highly dynamic, ranging from 2 copies per cell during stationary phase to ~1,200 copies per cell within 60 min of inoculation into fresh growth medium. Specificity of the assay for pre-rRNA was validated using rifampin and chloramphenicol, which are known inhibitors of pre-rRNA synthesis and processing, respectively. The DNA gyrase inhibitor, ciprofloxacin, was found to act similarly to rifampin; a decline in pre-rRNA was detectable within 15 min in ciprofloxacin-susceptible bacteria. Assays for pre-rRNA provide insight into cellular metabolism and are promising predictors of antibiotic susceptibility.

Design of Patchy Particles Using Quaternary Self-Assembled Monolayers

Binary and ternary self-assembled monolayers (SAMs) adsorbed on gold nanoparticles (NPs) have been previously studied for their propensity to form novel and unexpected patterns. The patterns found were shown to arise from a competition between immiscibilty of unlike surfactants and entropic gains due to length or other architectural differences between them. We investigate patterns self-assembled from quaternary monolayers on spherical nanoparticles. We perform simulations to study the effect of NP radius, degree of immiscibility between surfactants, length differences, and stoichiometry of the SAM on the formation of patterns. We report patterns analogous to binary and ternary cases, as well as some novel patterns specific to quaternary SAMs.

Design of patchy particles using ternary self-assembled monolayers

Binary and ternary self-assembled monolayers (SAMs) adsorbed on gold nanoparticles (NPs) have been previously studied for their propensity to form novel and unexpected patterns. The patterns found were shown to arise from a competition between immiscibilty of unlike surfactants and entropic gains due to length or other architectural differences between them. We investigate patterns self-assembled from quaternary monolayers on spherical nanoparticles. We perform simulations to study the effect of NP radius, degree of immiscibility between surfactants, length differences, and stoichiometry of the SAM on the formation of patterns. We report patterns analogous to binary and ternary cases, as well as some novel patterns specific to quaternary SAMs.

Ternary Monolayer Interfaces for Ultrasensitive and Direct Bioelectronic Detection of Nucleic Acids in Complex Matrices

AbstractElectrochemical nucleic acid biosensors have received considerable attention for the detection of biological agents. Control of the surface chemistry and coverage of the electrode transducer are essential for enhancing the performance of electrochemical DNA biosensors, and particularly for maximizing the hybridization efficiency and minimizing of nonspecific adsorption events. This review highlights recent advances and progress in the development of new surface chemistry approaches based on novel ternary DNA self‐assembled monolayer (SAM)‐interfaces using dithiols as new co‐adsorbents/‘backfillers’. The design and characterization of these powerful ternary SAM interfaces on different gold transducers are described, along with their implications to electrochemical biosensing of bioagents.

Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples

The development of rapid, low-cost and reliable diagnostic methods is crucial for the identification and treatment of many diseases. Screen-printed gold electrodes (Au/SPEs), coated with a ternary monolayer interface, involving hexanedithiol (HDT), a specific thiolated capture probe (SHCP), and 6-mercapto-1 hexanol (MCH) (SHCP/HDT/MCH) are shown here to offer direct and sensitive detection of nucleic acid hybridization events in untreated raw biological samples (serum, urine and crude bacterial lysate solutions). The composition of the ternary monolayer was modified and tailored to the surface of the Au/SPE. The resulting SHCP/HDT/MCH monolayer has demonstrated to be extremely useful for enhancing the performance of disposable nucleic acid sensors based on screen-printed electrodes. Compared to common SHCP/MCH binary interfaces, the new ternary self-assembled monolayer (SAM) resulted in a 10-fold improvement in the signal (S)-to-noise (N) ratio (S/N) for 1nM target DNA. The SHCP/HDT/MCH-modified Au/SPEs allowed the direct quantification of the target DNA down to 25pM (0.25fmol) and 100pM (1fmol) in undiluted/untreated serum and urine samples, respectively, and of 16S rRNA Escherichia coli (E. coli) corresponding to 3000CFUμL−1 in raw cell lysate samples. The new SAM-coated screen-printed electrodes also displayed favorable non-fouling properties after a 24h exposure to raw human serum and urine samples, offering great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests.

A Single‐Electrode, Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc-PNA biosensor (Fc: ferrocenyl, C(10)H(9)Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc-PNA capture probes were therefore synthesized by N-terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi-reversible redox processes of the Fc(0/+) redox couple with a significant difference in their electrochemical half-wave potentials of Delta E(1/2)=160 mV. A carefully designed biosensor interface, consisting of a ternary self-assembled monolayer (SAM) of the two C-terminal cysteine-tethered Fc-PNA capture probes and 6-mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear-cut electrochemical signal about the hybridization event. The discrimination of single-nucleotide polymorphism (SNP) was proven with a chosen single-mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual-potential sensor under real-life conditions.

Fabrication of Phase-Separated Multicomponent Self-Assembled Monolayers at Gold Nanoparticles Electrodeposited on Glassy Carbon Electrodes

This study aims at the fabrication of ternary self-assembled monolayers (ternary SAMs) composed of three kinds of thiol molecules with different lengths and different terminal function groups via a chemisorption on different domains of Au nanoparticles electrodeposited onto glassy carbon (GC) electrodes. The composition, domain size, and the surface concentration of each thiol molecule of the phase-separated ternary SAM are shown to be controllable. That is, the functionalization of the GC electrode (through the SAMs attachment) is composed of two consecutive processes, i.e., the electrodeposition of the Au nanoparticles followed by the fabrication of the SAM of a given thiol molecule. This electrodeposition-SAM formation process was repeated three times to fabricate phase-separated ternary SAM. The ternary SAM was composed of an unreactive methyl groups terminated long chain hydrophobic –alkylthiol (1-dodecanethiol, DDT), a reactive amino-group terminated thiol (11-amino undecanethiol, AUDT), and a reactive carboxylic acid group terminated short chain thiol (3-mercaptopropionic acid, MPA). The ternary SAM-modified GC electrode was further functionalized by confining an electroactive molecule having a redox center (i.e., aminoferrocene, AFc) via the covalent attachment to the carboxylic acid group of the MPA moiety of the ternary SAM. The molecular-level control of the structure of the SAMs formed on the different Au nanoparticles domains is a promising criterion that could be utilized to fabricate multifunctional nanoparticles-based electrodes, for example, to analyze several bioactive species.

Concentration-dependent switching of the mode of phase separation in ternary self-assembled monolayers of 2-mercaptoethane sulfonic acid, 2-aminoethanethiol and 1-dodecanethiol on Au(1 1 1)

Abstract Unusually strong apparent surface activity of the pair of 2-mercaptoethane sulfonic acid (MES) and 2-aminoethanethiol (AET) in forming a phase-separated ternary self-assembled monolayer (SAM) composed of MES, AET, and 1-dodecanethiol (DDeT) has been investigated with changing the conditions for preparing the SAM by coadsorption of the thiols from an ethanolic solution. When the total concentration of the thiols, c total , is 1 × 10 −3 mol dm −3 or higher, the MES–AET pair forms electrostatically stabilized domains from an early stage of the adsorption after the immersion of a gold substrate in the bathing ethanol solution. The total area of the MES–AET domains does not change with time for longer immersion. The adsorption of DDeT is significantly weaker than the MES–AET pair under such conditions, despite the fact that the intrinsic surface activity of DDeT is greater than those of MES and AET. DDeT-rich islands of typically a few nanometer across spread in the SAM and the size and the distribution of the islands are invariant over a week. In contrast, when c total is lower than 0.1 × 10 −3 mol dm −3 , the adsorptivity of DDeT is stronger than that of the MES–AET pair, and MES and AET initially adsorbed are gradually replaced with DDeT at longer immersion time. This switching of the mode of phase separation with the change in c total suggests the crucial roles of the electrostatic interaction of MES and AET in the course of the formation of the SAM and of the hydrophilic barrier formed on the domains to prevent the replacement by hydrophobic DDeT molecules.

Phase Separation of Ternary Self-Assembled Monolayers into Hydrophobic 1-Dodecanethiol Domains and Electrostatically Stabilized Hydrophilic Domains Composed of 2-Aminoethanethiol and 2-Mercaptoethanesulfonic Acid on Au(111)

Ternary self-assembled monolayers (SAM) composed of 2-aminoethanethiol (AET), 2-mercaptoethanesulfonic acid (MES), and 1-dodecanethiol (DDeT) form two types of domains as if it were a two-component SAM: DDeT-rich hydrophobic domains and electrostatically stabilized hydrophilic domains composed of MES and AET on Au(111). MES and AET behave virtually as a single surface-active species. Two distinct reductive desorption peaks in cyclic voltammograms (CV) and binarized images of scanning tunneling microscopy clearly show nanometer scale, yet macroscopically distinguishable, phase separation over a wide range of the mixing ratio of DDeT and MES-AET in the bathing solution. X-ray photoelectron spectroscopy measurements indicate that the ratio of MES to AET in the hydrophilic domains is unity and that both terminal groups are in the charged states, that is, the sulfonate group and the ammonium group. With decreasing the total concentration of the thiols, the mole fraction of DDeT in the bathing solution at which the surface coverage of MES-AET domains is equal to that of DDeT domains dramatically decreases. This suggests that the adsorption kinetics plays a crucial role in the formation of the domains structure.

Direct Electrochemical Transduction of Biorecognition at Viologen-Containing Monolayer Surfaces

We describe a new, direct electrochemical means of detecting biorecognition at modified electrodes. Ternary self-assembled monolayers (SAMs) were formed on gold electrodes by coadsorption of biotin, viologen, and oligo(ethylene glycol)-functionalized alkanethiols. These SAMs exhibit electroactivity of the viologen moieties, biospecificity of the biotin moieties, and inhibition of nonspecific adsorption by the oligo(ethylene glycol) moieties. Changes in electrochemical behavior of the immobilized viologen observed by cyclic voltammetry accompanying biospecific adsorption of an anti-biotin antibody to biotin can be used to transduce biorecognition on the surface rapidly and reproducibly. Surface plasmon resonance measurements are consistent with the electrochemical responses that result from the partially reversible adsorption of the antibody to the SAM surface.