Thiol-modified Single-stranded DNA Research Articles

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

Label-free electrochemical biosensor with magnetically induced self-assembly for the detection of cancer antigen 125

Magnetically induced self-assembly technology was used to construct a label-free electrochemical biosensor based on magnetic Mg0.5Cu0.5Fe2O4-Au nanocomposites for the sensitive detection of cancer antigen 125 (CA125). To aim for this, Mg0.5Cu0.5Fe2O4 nanoparticles were first prepared via the rapid combustion process. The average diameter of the nanoparticles calcined at 800 °C with the absolute ethanol volume of 20 mL was about 169.3 nm. Then Au was used for the surface modification by NaBH4 reduction reaction approach. The magnetic glassy carbon electrode (MGCE) was modified by Mg0.5Cu0.5Fe2O4-Au via a magnetic induction self-assembly process. The reduced thiol-modified single-stranded DNA was attached to the Mg0.5Cu0.5Fe2O4-Au nanocomposites by Au-S bonds without any coupling agent. CA125 antigen was grabbed directly by its aptamer DNA due to its specific identification with the aptamer. Finally, the modified electrodes were blocked with BSA and then characterized. Finally, DPV analysis was used for CA125 detection, the novel fabricated biosensor demonstrated good detection properties for CA125 with a linear range of 5–125 U/mL and a detection limit of 4.4 U/mL. The results showed that the aptamer sensor had good specificity, repeatability, and stability. The feasibility of our sensor for the determination of CA125 was also demonstrated by measuring CA125 levels in serum using the Roche gold-standard instrument of the People’s Hospital of Danyang as the reference value. The recoveries of real serum samples were 94.65–101.71%, and RSDs were 1.26–4.65%. Moreover, the surface of the electrode could be cleaned and reused by magnetic separation, greatly reducing the cost and providing the possibility for a point-of-care test (POCT). This work demonstrated a new strategy for integrating both nanostructures and biocompatibility to build advanced cancer biomarker sensors with wide applications.

Design of a new electrochemical aptasensor based on screen printed carbon electrode modified with gold nanoparticles for the detection of fumonisin B1 in maize flour

A new aptasensor for detecting fumonisin B1 (FB1) in the maize samples was developed based on DNA- aptamer recognition and electrochemical technique. A thiol-modified single-stranded DNA (ss-HSDNA) was immobilized on a screen printed carbon electrode (SPCE) electrodeposited by gold nanoparticles (AuNPs). The morphology and structure of SPCE and AuNPs/SPCE were evaluated via scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The SEM results demonstrated that the SPCE had a flat sheet-like structure, and the AuNPs were homogeneously electrodeposited on the SPCE. Cyclic voltammetry (CV) experiments in the [Fe(CN)6]− 3/− 4 solution were conducted to investigate each step of electrode modification as well as aptasensor performance. Aptamer-FB1 interaction prevented the electron transfer permitting the determination of FB1 in the range of 0.5–500 ng/mL with a low detection limit (0.14 ng/mL). The designed aptasensor was also shown high selectivity, acceptable repeatability and reproducibility, good long-term stability, and excellent recovery. Furthermore, there was a strong correlation between the findings achieved via the designed aptasensor and high performance liquid chromatography (HPLC). Therefore, a simple construction process and satisfactory electrochemical performance of the proposed aptasensor have a great potential for the detection of FB1 in maize samples.

Flexible ITO Electrode With Gold Nanostructures for Femtomolar DNA Hybridization Detection

In this article, we report detection of DNA hybridization using a gold nanostructure coated flexible indium tin oxide (ITO) working electrode, aiming at exploring alternate materials for developing low-cost light-weight sensing platforms in the future. Herein, gold nanostructures were electrodeposited onto the flexible ITO substrate with HAuCl4 precursor solution. To prepare the bioelectrode for DNA sensing, thiol-modified single-stranded DNA probes specific to the BRCA1 gene were attached to the nanostructured gold surface via self-assembly. When subjected to hybridization detection in a wide concentration range of 1fM1M, the bioelectrode resulted in a limiting detection of 0.255fM (calculated with 3.3 method), with a sensitivity of 10.94(Amol)cm2. In order to estimate the efficiency of the sensor, we have performed interference analysis in the presence of a noncomplementary target DNA and human serum albumin. Furthermore, the reproducibility of the biosensor was verified using a blind testing protocol.

A novel biomimetic olfactory cell-based biosensor with DNA-directed site-specific immobilization of cells on a microelectrode array

The coupling of cells with transducers is a key step to develop cell-based biosensors in order to meet the specific performance requirements for biomedical applications. This study describes a novel biomimetic olfactory cell-based biosensor by the employment of a DNA-directed site-specific cell immobilization method, which can achieve controllable and high-efficient coupling between olfactory cells and the microelectrode array (MEA) chip. A pair of complementary thiol-modified single-stranded DNA (ssDNA) was designed and synthesized. One of them was covalently attached to the plasma membrane of olfactory cells that were isolated from rats. The other one was used as ssDNA probes and immobilized on the gold surface of microelectrodes on MEA chip. Based on the mechanism of complementary ssDNA hybridization, olfactory cells were site-specifically immobilized on the surface of microelectrodes. The distribution of olfactory cells on the surface of MEA chip was controlled by the pattern of ssDNA probes on the sensor surface. The fluorescent staining results indicate that olfactory cells were site-specifically coupled with the microelectrode array on the surface of a MEA chip, which formed a desirable olfactory cell array. In addition, extracellular recordings from the coupled olfactory cells demonstrate that microelectrodes can efficiently monitor the membrane potential changes originated from olfactory cells in response to odorant stimulations. This biomimetic olfactory cell-based biosensor can not only be used for the detection of odorant molecules, but also be applied for the research on the olfactory signal transduction mechanisms. This DNA-directed cell immobilization method provides a novel approach for controllable cell immobilization on solid support and consequently, contributes to the development of cell micro arrays for cell-based biosensors.

Detection of mRNA from Escherichia coli in drinking water on nanostructured polymeric surfaces using liquid crystals

In this study, we demonstrate the detection of mRNA from Escherichia coli in drinking water using thermotropic liquid crystals (LCs). After hybridization of complementary mRNA with the single-stranded DNA immobilized on a polymer substrate containing periodic sinusoidal wave patterns, the orientation of LCs transits from a uniform to a non-uniform state, thereby inducing a change in the optical response of LCs. The periodic sinusoidal features of the polymer substrate are obtained through buckling the poly-(dimethylsiloxane) slide on a cylindrical surface, followed by replicating the associated relief structures on a poly-(urethaneacrylate) surface, where a film of gold was deposited. Then, thiol-modified single-stranded DNA was functionalized on the gold film as an mRNA receptor. The formation of mRNA–single-stranded DNA complex, which covers the sinusoidal nanostructures on the surface, induces the orientational transition of LCs. This result indicates that LCs can be used to report the specific hybridization of mRNA with single-stranded DNA, which holds promise for the sensitive and label-free detection of viable bacterial pathogens in drinking water.

Label-free detection of protein–DNA interactions using electrochemical impedance spectroscopy

In this work, the applicability of an impedimetric DNA sensor has been investigated for the detection of protein–DNA interactions. The sensor is based on short thiol-modified single-stranded DNA, which is chemisorbed to gold chip electrodes. In the presence of the redox system ferri-/ferrocyanide impedance measurements show an increase in charge transfer resistance after immobilization and hybridization of ssDNA to the sensor surface. The use of a longer capture oligonucleotide (a 25-mer instead of an 18-mer) results in a decreasing probe concentration on the surface. Furthermore it causes an increase of the charge transfer resistance for both ssDNA and dsDNA. The hybridization event, however, can be detected with a similar sensitivity compared to an 18-mer (with the same surface concentration) and allows a good discrimination between ssDNA and dsDNA. This electrode system is used to follow an enzyme reaction on the surface electrochemically. The cleavage of a double-stranded DNA by restriction endonuclease BamHI could be verified by cyclic voltammetry and impedance spectroscopy. The investigations are performed in dithiothreitol (DTT) free buffer solution since the incubation with DTT results in an alteration of the surface impedance. The sequence specific DNA-binding of the transcription factor NF-κB p50 is found to cause a decrease in charge transfer resistance. The signal change is concentration dependent and occurs due to a neutralization effect of the negatively charged DNA backbone.

Exploration of the specific structural characteristics of thiol-modified single-stranded DNA self-assembled monolayers on gold by a simple model

This paper proposed a simple hexagonal model to explore the specific structural characteristics of thiol-modified single-stranded DNA (ss-DNA) self-assembled monolayers (SAMs) on gold substrate. The calibrated gyration diameter d ′ g ( d ′ g = r d g ) was used to quantify the size of ss-DNA molecules on gold by introducing a calibrating factor r, where d g was ss-DNA gyration diameter in solution. Based on the model, the interfacial parameters of ss-DNA-SAMs on gold assembled under different ionic strength were obtained theoretically. The ss-DNA-SAMs were assembled on gold under different concentrations of C NaCl and six important electrochemical parameters were used to validate the model experimentally, which include surface coverage ( Γ m), interfacial capacitance ( C), phase angle ( Ф 1 Hz ), ions transfer resistance ( R it * ), current density difference (Δ j) and charge transfer resistance ( R ct) from chronocoulometry ( CC), cyclic voltammetry ( CV) and electrochemical impedance spectroscopy ( EIS). Three main aspects were included in this paper: (1) construction of a simple hexagonal model to describe the specific structural characteristics of ss-DNA-SAMs on gold; (2) calculation of the calibrating factor r by CC experiments and several important conclusions from the simple model; and (3) confirmation of the simple model by our experimental results and literature reports. The simple model may provide an important reference for optimizing the design of DNA sensor.

Direct Detection of DNA and DNA-Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA-ligand interactions. The sensor system consists of thiol-modified single-stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri-/ferrocyanide show an increase in charge transfer resistance (Rct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm2 ssDNA. The relative change in Rct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5- to 4.5-fold. Impedimetric detection of binding events of a metal-intercalator ([Ru(phen)3]2+) and a groove binder (spermine) to double-stranded DNA is demonstrated. Binding of [Ru(phen)3]2+ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single-stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single-stranded DNA. The specific binding of an anti-dsDNA antibody to the dsDNA-modified electrode surface decreases in contrast the interfacial impedance.

Highly Sensitive Resonance Scattering Detection of DNA Hybridization Using Aptamer-Modified Gold Nanopaticle as Catalyst

Gold nanoparticle particles in size of 10 nm were used to label the thiol-modified single-stranded DNA aptamer (SH-ssDNA) to obtain an aptamer-modified gold nanoparticle probe (AussDNA) for target DNA (tDNA). In pH 7.4 NaH2PO4–Na2HPO4 buffer solution, the hybridization reaction between AussDNA and tDNA took place to form larger aptamer-modified gold nanoparticle cluster complex. The excess aptamer-modified gold nanoparticle probe in the supernatant solutions was obtained by centrifuging and can be used as nanocatalyst for the 0.276 mmol/L CuSO4-65.4 mmol/L potassium-sodium tartrate-0.37 mmol/L glucose system at 70 °C. The cubic Cu2O particles generated by the nanocatalytic reducing exhibit a strong resonance scattering (RS) peak at 620 nm. In the selected conditions, the RS intensity at 620 nm decreased with addition of tDNA, and the decreased intensity ΔI 620 nm is proportional to tDNA concentration (C tDNA) from 0.12 to 72 pM, with regress equation of ΔI 620 nm = 1.29C tDNA + 4.05, correlation coefficient of 0.9917, and detection limit of 0.084 pM tDNA.

Hybrid Surface Platform for the Simultaneous Detection of Proteins and DNAs Using a Surface Plasmon Resonance Imaging Sensor

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.

Binary monolayers of single-stranded oligonucleotides and blocking agent for hybridisation

Abstract The hybridisation of thiol-modified single-stranded DNA (SH-ssDNA) embedded in lipoate or hydrophilic polymer monolayers on gold have been studied through the technique of surface plasmon resonance (SPR). SH-ssDNA and the blocking agents were co-adsorbed on gold from the same solution or the surface was post-treated with the blocking agents. The assembling process is allowed to take place for only 10 min. Binding of non-complementary and complementary DNA is dependent both on the blocking agent and on the assembling method used. The lowest non-complementary binding and the highest complementary binding of DNA was obtained with a layer assembled from a binary solution of SH-ssDNA and the lipoate blocking agent.

Hybridisation of surface-immobilised single-stranded oligonucleotides and polymer monitored by surface plasmon resonance

We have investigated the hybridisation of thiol-modified single-stranded DNA embedded in a polyacrylamide layer through the technique of surface plasmon resonance (SPR). Kinetic studies were carried out by two different immobilisation methods: (a) SH-ssDNA was firstly attached on gold and the remaining free space was filled with polymer and (b) SH-ssDNA and the polymer was attached onto the surface from the same solution. The immobilisation methods were compared for various concentrations of SH-ssDNA. Hybridisation was dependent on both the immobilisation method and the concentration of the components. The highest hybridisation was obtained when SH-ssDNA and the polymer was immobilised from the same solution at low SH-ssDNA concentration or when high concentrations of oligos were spread onto the surface and the surface was post-treated with polymer. The target response corresponded to a surface coverage of 100+/-15 ng/cm2. The same surface coverage on hybridisation was also obtained when low concentration of SH-ssDNA and polymer was attached onto the surface from the same solution. The non-specific binding of sample DNA was very low at optimal concentrations due to the polymer and the hybridisation was linearly dependent on target concentration.