Effects of gold nanoparticle and electrode surface properties on electrocatalytic silver deposition for electrochemical DNA hybridization detection

In this paper we report the catalytic effects of various gold nanoparticles for silver electrodeposition on indium tin oxide (ITO)-based electrodes, and successfully apply this methodology for signal amplification of the hybridization assay. The most widely used gold nanoparticle-based hybridization indicators all promote silver electrodeposition on the bare ITO electrodes, with decreasing catalytic capability in order of 10 nm gold, DNA probe-10 nm gold conjugate, streptavidin-5 nm gold, and streptavidin-10 nm gold. Of greater importance, these electrocatalytic characteristics are affected by any surface modifications of the electrode surfaces. This is illustrated by coating the ITO with an electroconducting polymer, poly(2-aminobenzoic acid)(PABA), as well as avidin molecules, which are promising immobilization platforms for DNA biosensors. The catalytic silver electrodeposition of the gold nanoparticles on the PABA-coated ITO surfaces resembles that on the bare surfaces. With avidin covalently bound to the PABA, it is interesting to note that the changes in electrocatalytic performance vary for different types of gold nanoparticles. For the streptavidin-5 nm gold, the silver electrodeposition profile is unaffected by the presence of the avidin layer, whereas for both the 10 nm Au and DNA probe-10 nm gold conjugate, the deposition profiles are suppressed. The streptavidin-5 nm gold is employed as the hybridization indicator, with avidin-modified (via PABA) ITO electrode as the immobilization platform, to enable signal amplification by the silver electrodeposition process. Under the conditions, this detection strategy offers a signal-to-noise ratio of 20. We believe that this protocol has great potential for simple, reproducible, highly selective and sensitive DNA detection on fully integrated microdevices in clinical diagnostics and environmental monitoring applications.     

Label-free electrochemical impedance sensing of DNA hybridization based on functionalized graphene sheets

Negative-charge change and conformation transition upon DNA immobilization and hybridization on functionalized graphene sheets were monitored by the EIS technique and adopted as the signal for label-free electrochemical DNA hybridization detection.     

Label-free PSA electrochemical determination by seed-mediated electrochemically-deposited gold nanoparticles on an FTO electrode

Journal of Solid State Electrochemistry - A facile and sensitive approach is introduced to precisely determine trace amounts of prostate specific antigen (PSA) by gold nanostructures deposited on...     

Label-free electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, polythionine, and graphene

We describe the fabrication of a sensitive label-free electrochemical biosensor for the determination of sequence-specific target DNA. It is based on a glassy carbon electrode (GCE) modified with graphene, gold nanoparticles (Au-NPs), and polythionine (pThion). Thionine was firstly electropolymerized on the surface of the GCE that was modified with graphene by cyclic voltammetry. The Au-NPs were subsequently deposited on the surface of the pThion/graphene composite film by adsorption. Scanning electron microscopy and electrochemical methods were used to investigate the assembly process. Differential pulse voltammetry was employed to monitor the hybridization of DNA by measuring the changes in the peak current of pThion. Under optimal conditions, the decline of the peak current is linearly related to the logarithm of the concentration of the target DNA in the range from 0.1 pM to 10 nM, with a detection limit of 35 fM (at an S/N of 3). The biosensor exhibits good selectivity, acceptable stability and reproducibility.

Chemical control of electrode functionalization for detection of DNA hybridization by electrochemical impedance spectroscopy

We report sensitive label-free detection of DNA oligonucleotide sequences using ac impedance measurements. The surface attachment chemistry is critical, and using mixed self-assembled monolayers on a gold electrode results in much better performance than homogeneous self-assembled monolayers. Contrary to expectations, binding of the target sequence reduces rather than increases the charge transfer resistance. Similar behavior is observed on indium tin oxide electrodes, and we ascribe it to the hydrophilicity and rigidity of the DNA duplex that cause it to reside further from the electrode surface and facilitate the approach of negatively charged redox moieties to the interface.     

Magnetically-induced solid-state electrochemical detection of DNA hybridization

A magnetic triggering of a solid-state electrical transduction of DNA hybridization is described. Positioning of an external magnet below the thick-film electrode attracts the DNA/particle network and enables the solid-state electrochemical stripping detection of the silver tracer. TEM imaging indicates that the hybridization event results in a three-dimensional aggregate structure in which duplex segments link the metal nanoparticles and magnetic spheres, and that most of this assembly is covered with the silver precipitate. This leads to a direct contact of the metal tag with the surface (in connection to the magnetic collection) and enables the solid-state electrochemical transduction (without prior dissolution and subsequent electrodeposition of the metal), using oxidative dissolution of the silver tracer. No such aggregates (and hence magnetic "collection") are observed in the presence of noncomplementary DNA, that is, without the linking hybrid. The new method couples high sensitivity of silver-amplified assays with effective discrimination against excess of closely related nucleotide sequences (including single-base imperfections). Such direct electrical detection of DNA/metal-particle assemblies can bring new capabilities to the detection of DNA hybridization, and could be applied to other bioaffinity assays.     

Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection

A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH) for covalent DNA immobilization and enhanced hybridization detection is described. The MWNTs-COOH-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides with the 5'-amino group were covalently bonded to the carboxyl group of carbon nanotubes. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using an electroactive intercalator daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity. This is the first application of carbon nanotubes to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.     

Cadmium sulfide nanocluster-based electrochemical stripping detection of DNA hybridization

A novel, sensitive electrochemical DNA hybridization detection assay, using cadmium sulfide (CdS) nanoclusters as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the CdS nanocluster oligonucleotide DNA probe, followed by the dissolution of the CdS nanoclusters anchored on the hybrids and the indirect determination of the dissolved cadmium ions by sensitive anodic stripping voltammetry (ASV) at a mercury-coated glassy carbon electrode (GCE). The results showed that only a complementary sequence could form a double-stranded dsDNA-CdS with the DNA probe and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. The combination of the large number of cadmium ions released from each dsDNA hybrid with the remarkable sensitivity of the electrochemical stripping analysis for cadmium at mercury-film GCE allows detection at levels as low as 0.2 pmol L(-1) of the complementary sequence of DNA.     

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.     

Modification of Escherichia coli single-stranded DNA binding protein with gold nanoparticles for electrochemical detection of DNA hybridization

The unique binding event between Escherichia coli single-stranded DNA binding protein (SSB) and single-stranded oligonucleotides conjugated to gold (Au) nanoparticles is utilized for the electrochemical detection of DNA hybridization. SSB was attached onto a self-assembled monolayer (SAM) of single-stranded oligonucleotide modified Au nanoparticle, and the resulting Au-tagged SSB was used as the hybridization label. Changes in the Au oxidation signal was monitored upon binding of Au tagged SSB to probe and hybrid on the electrode surface. The amplified oxidation signal of Au nanoparticles provided a detection limit of 2.17 pM target DNA, which can be applied to genetic diagnosis applications. This work presented here has important implications with regard to combining a biological binding event between a protein and DNA with a solid transducer and metal nanoparticles.     

Label-free immunosensing of microcystin-LR using a gold electrode modified with gold nanoparticles

The hepatotoxic microcystins, especially microcystin?CLR (MC?CLR), are causing serious problems to public health and fisheries. We describe here a label-free amperometric immunosensor for rapid determination of MC?CLR in water sample. The sensor was prepared by immobilizing antibody on a gold electrode coated with L-cysteine-modified gold nanoparticles. The stepwise self-assembly of the immunosensor was monitored and characterized by means of electrochemical impedance spectroscopy and differential pulse voltammetry. A 0.60?mmol L^?1 solution of hydroquinone was used as the electron mediator. The immunosensor was incubated with MC?CLR at 25?°C for 20?min, upon which the differential pulse voltammetric current changed linearly over the concentration range from 0.05 to 15.00???g L^?1, with a detection limit of 20?ng L^?1. The developed biosensor was used to determine MC?CLR in spiked crude algae samples. The recovery was in the range from 95.6 to 105%. This method is simple, economical and efficient, this making it potentially suitable for field analysis of MC-LR in crude algae and water samples.

Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV–vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10⁻¹³ to 4.5 × 10⁻⁶ mol/L. The detection limit (3σ) of this method has decreased to pico-molar level.     

Label-free detection of DNA molecules on the dendron based self-assembled monolayer by electrochemical impedance spectroscopy

We report preparation of a novel platform for effective DNA hybridization and its application to the detection of single mismatched DNA. Cone-shaped dendrimer molecules have been immobilized on the gold surface at equidistance, 3.1 nm, from each other with a probe DNA molecule attached to the top of each dendrimer so that enough space would be secured for effective hybridization. This arrangement allows each probe DNA molecule to form a natural DNA double helix upon hybridization with a target DNA molecule. The single nucleotide polymorphism at either the central or end position of the 25-mer target DNA has been shown to be effectively discriminated against on this platform from each other as well as from a complementary DNA by electrochemical impedance measurements. We also report adverse effects exerted by probe ions, Fe(CN)₆^3−/4−, on DNA hybridization reactions. The significance of the results for the use in DNA analysis is discussed.     

Label-free DNA hybridization probe based on a conducting polymer

A new approach to the realization of the electrochemical DNA hybridization probe is described. It is based on the exchange of chloride ion between the polypyrrole layer and the buffer. The shape of the cyclic voltammogram is modulated by the negative charge density at this interface, resulting from the immobilized target DNA. The negative charge density increases when the complementary DNA hybridizes with the probe DNA. The hybridization event can be clearly seen in the cyclic voltammogram before and after the addition of the probe DNA. The immobilization is accomplished via the Mg2+ bridging complex between phosphonic acid groups of the poly[2,5-dithienyl-(N-3-phosphorylpropyl)pyrrole] grafted at the polypyrrole surface and the phosphate groups of the target DNA.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.     

Label-free "digital detection" of single-molecule DNA hybridization with a single electron transistor

Here we present a novel assay that eliminates fluorescent labels and enables "digital detection" of single-molecule DNA hybridization in complex matrixes with greatly simplified protocols. Electronic coupling of the binding state of a single oligonucleotide to the quantum dot (QD) of a single electron transistor (SET) affords direct observation of binding events in real-time via "molecular gating". The change of electrostatic charge associated with the molecular capture is used in lieu of a gate electrode to modulate the SET conductivity. Target oligos containing base mismatches do not elicit SET response under 0.1X SSC at room temperature nor do changes in ionic strength or pH. Furthermore, hybridization is detected even in optically inaccessible matrixes such as serum or quanidinium thiocyanate lysis buffer.