Enhancement of Electrocatalytic Activity of DNA-Conjugated Gold Nanoparticles and Its Application to DNA Detection

Hydrazine electrooxidation readily occurs on bare Au nanoparticles (AuNPs), whereas it does not on DNA-conjugated AuNPs. Thus, when DNA-conjugated AuNPs are used as electrocatalytic labels in electrochemical DNA detection, anodic current of hydrazine is not easily observed within the potential window because of the high overpotential caused by the slow electron-transfer kinetics on DNA-conjugated AuNPs as well as the slow electron tunneling between the AuNP and the indium−tin oxide (ITO) electrode. NaBH4 treatment significantly enhances the electrocatalytic activity of DNA-conjugated AuNPs. This enhancement substantially decreases the overpotential caused by the slow electron-transfer kinetics, and the anodic current of hydrazine can be measured within the potential window if the distance between the AuNP and the ITO electrode is not too large. The enhancement with NaBH4 treatment allows high signal current, and the low intrinsic electrocatalytic activity of ITO electrodes allows low background current. The high signal-to-background ratio enables us to detect 1 fM target DNA without target amplification or enzymatic signal amplification. The ultrasensitive detection using versatile AuNP and simple chemical treatment is practically appealing

Ultrasensitive Detection of DNA in Diluted Serum Using NaBH₄ Electrooxidation Mediated by [Ru(NH₃)₆]³⁺ at Indium−Tin Oxide Electrodes

There is a crucial need for simple and highly sensitive techniques to detect DNA in complicated biological samples such as serum. Here we present an ultrasensitive electrochemical DNA sensor using (i) single DNA hybridization with peptide nucleic acid (PNA), (ii) selective binding of [Ru(NH3)6]3+ to hybridized DNA, (iii) fast NaBH4 electrooxidation mediated by [Ru(NH3)6]3+, and (iv) low background currents of NaBH4 at indium−tin oxide (ITO) electrodes. The [RuIII(NH3)5NH2]2+ formed from [RuIII(NH3)6]3+ in borate buffer (pH 11.0) is readily electrooxidized to both [RuIV(NH3)5NH2]3+ and Ru complex with a higher oxidation state. In the absence of [Ru(NH3)6]3+ bound to the DNA-sensing ITO electrodes, the oxidation currents of NaBH4 are very low. However, in the presence of [Ru(NH3)6]3+, the oxidation currents of NaBH4 are highly enhanced due to electron mediation of the oxidized Ru complexes. The significant enhancement in the electrocatalytic activity of sensing electrodes after [Ru(NH3)6]3+ binding facilitates to obtain high signal-to-background ratios. PNA and ethylenediamine on DNA-sensing electrodes significantly decrease [Ru(NH3)6]3+ binding, also allowing for high signal-to-background ratios. The oxidation charges of NaBH4 obtained from chronocoulometry are highly reproducible. All combined effects enable the detection of DNA with a detection limit of 1 fM in ten-fold diluted human serum. The simple and fast detection procedure and the ultrasensitivity make this approach highly promising for practical DNA detection

A Nanocatalyst-Based Assay for Proteins: DNA-Free Ultrasensitive Electrochemical Detection Using Catalytic Reduction of <i>p</i>-Nitrophenol by Gold-Nanoparticle Labels

This communication reports a nanocatalyst-based electrochemical assay for proteins. Ultrasensitive detection has been achieved by signal amplification combined with noise reduction:  the signal is amplified both by the catalytic reduction of p-nitrophenol to p-aminophenol by gold-nanocatalyst labels and by the chemical reduction of p-quinone imine to p-aminophenol by NaBH4; the noise is reduced by employing an indium tin oxide electrode modified with a ferrocenyl-tethered dendrimer and a hydrophilic immunosensing layer

Electrochemical Immunosensor Using <i>p</i>-Aminophenol Redox Cycling by Hydrazine Combined with a Low Background Current

Signal amplification and noise reduction are crucial for obtaining low detection limits in biosensors. Here, we present an electrochemical immunosensor in which the signal amplification is achieved using p-aminophenol (AP) redox cycling by hydrazine, and the noise level is reduced by implementing a low background current. The redox cycling is obtained in a simple one-electrode, one-enzyme format. In a sandwich-type heterogeneous immunosensor for mouse IgG, an alkaline phosphatase label converts p-aminophenyl phosphate into AP for 10 min. This generated AP is electrooxidized at an indium tin oxide (ITO) electrode modified with a partially ferrocenyl-tethered dendrimer (Fc-D). The oxidized product, p-quinone imine (QI), is reduced back to AP by hydrazine, and then AP is electrooxidized again to QI, resulting in redox cycling. Moreover, hydrazine protects AP from oxidation by air, enabling long incubation times. The small amount of ferrocene in a 0.5% Fc-D-modified ITO electrode, where 0.5% represents the ratio of ferrocene groups to dendrimer amines, results in a low background current, and this electrode exhibits high electron-mediating activity for AP oxidation. Moreover, there is insignificant hydrazine electrooxidation on this electrode, which also results in a low background current. The detection limit of the immunosensor using a 0.5% Fc-D-modified electrode is 2 orders of magnitude lower than that of a 20% Fc-D-modified electrode (10 pg/mL vs 1 ng/mL). Furthermore, the presence of hydrazine reduces the detection limit by an additional 2 orders of magnitude (100 fg/mL vs 10 pg/mL). These results indicate that the occurrence of redox cycling combined with a low background current yields an electrochemical immunosensor with a very low detection limit (100 fg/mL). Mouse IgG could be detected at concentrations ranging from 100 fg/mL to 100 μg/mL (i.e., 9 orders of magnitude) in a single assay

DNA Clutch Probes for Circulating Tumor DNA Analysis

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification

Proximal Bacterial Lysis and Detection in Nanoliter Wells Using Electrochemistry

Rapid and direct genetic analysis of low numbers of bacteria using chip-based sensors is limited by the slow diffusion of mRNA molecules. Long incubation times are required in dilute solutions in order to collect a sufficient number of molecules at the sensor surface to generate a detectable signal. To overcome this barrier here we present an integrated device that leverages electrochemistry-driven lysis less than 50 μm away from electrochemical nucleic acid sensors to overcome this barrier. Released intracellular mRNA can diffuse the short distance to the sensors within minutes, enabling rapid and sensitive detection. We validate this strategy through direct lysis and detection of E. coli mRNA at concentrations as low as 0.4 CFU/μL in 2 min, a clinically relevant combination of speed and sensitivity for a sample-to-answer molecular analysis approach

Comparison of the Nonspecific Binding of DNA-Conjugated Gold Nanoparticles between Polymeric and Monomeric Self-Assembled Monolayers

The nonspecific binding of DNA-conjugated gold nanoparticles (AuNPs) to solid surfaces is more difficult to control than that of DNA molecules due to the more attractive interactions from the large number of DNA molecules per AuNP. This paper reports that the polymeric self-assembled monolayers (SAMs) formed on indium−tin oxide (ITO) electrodes significantly inhibit the nonspecific binding of DNA-conjugated AuNPs. The random copolymers used to prepare the polymeric SAMs consist of three functional parts: an ITO-reactive silane group, a DNA-blocking poly(ethylene glycol) (PEG) group, and an amine-reactive N-acryloxysuccinimide group. In order to compare the polymeric SAMs with various monomeric SAMs, the relative nonspecific binding of the DNA-conjugated AuNPs to the ITO electrodes modified with (3-aminopropyl)triethoxysilane (APTES), 3-aminopropylphosphonic acid, 3-phosphonopropionic acid, or 11-phosphonoundecanoic acid is examined by measuring the electrocatalytic anodic current of hydrazine caused by the nonspecifically absorbed AuNPs and by counting the AuNPs adsorbed onto modified ITO electrodes. Carboxylic-acid-terminated and amine-terminated monomeric SAMs cause high levels of nonspecific binding of DNA-conjugated AuNPs. The monomeric SAM modified with the carboxylic-acid-terminated poly(amidoamine) dendrimer shows low levels of nonspecific binding (2.0% nonspecific binding relative to APTES) due to the high surface density of the negative charge. The simply prepared polymeric SAM produces the lowest level of nonspecific binding (0.8% nonspecific binding relative to APTES), resulting from the combined effect of (i) DNA-blocking PEG and carboxylic acid groups and (ii) dense polymeric SAMs. Therefore, thin and dense polymeric SAMs may be effective in electrochemical detection and easy DNA immobilization along with low levels of nonspecific binding

Nanocatalyst-Based Assay Using DNA-Conjugated Au Nanoparticles for Electrochemical DNA Detection

Compared to enzymes, Au nanocatalysts show better long-term stability and are more easily prepared. Au nanoparticles (AuNPs) are used as catalytic labels to achieve ultrasensitive DNA detection via fast catalytic reactions. In addition, magnetic beads (MBs) are employed to permit low nonspecific binding of DNA-conjugated AuNPs and to minimize the electrocatalytic current of AuNPs as well as to take advantage of easy magnetic separation. In a sandwich-type electrochemical sensor, capture-probe-conjugated MBs and an indium−tin oxide electrode modified with a partially ferrocene-modified dendrimer act as the target-binding surface and the signal-generating surface, respectively. A thiolated detection-probe-conjugated AuNP exhibits a high level of unblocked active sites and permits the easy access of p-nitrophenol and NaBH4 to these sites. Electroactive p-aminophenol is generated at these sites and is then electrooxidized to p-quinoneimine at the electrode. The p-aminophenol redox cycling by NaBH4 offers large signal amplification. The nonspecific binding of detection-probe-conjugated AuNPs is lowered by washing DNA-linked MB−AuNP assemblies with a formamide-containing solution, and the electrocatalytic oxidation of NaBH4 by AuNPs is minimized because long-range electron transfer between the electrode and the AuNPs bound to MBs is not feasible. The high signal amplification and low background current enable the detection of 1 fM target DNA

High-Curvature Nanostructuring Enhances Probe Display for Biomolecular Detection

High-curvature electrodes facilitate rapid and sensitive detection of a broad class of molecular analytes. These sensors have reached detection limits not attained using bulk macroscale materials. It has been proposed that immobilized DNA probes are displayed at a high deflection angle on the sensor surface, which allows greater accessibility and more efficient hybridization. Here we report the first use of all-atom molecular dynamics simulations coupled with electrochemical experiments to explore the dynamics of single-stranded DNA immobilized on high-curvature versus flat surfaces. We find that high-curvature structures suppress DNA probe aggregation among adjacent probes. This results in conformations that are more freely accessed by target molecules. The effect observed is amplified in the presence of highly charged cations commonly used in electrochemical biosensing. The results of the simulations agree with experiments that measure the degree of hybridization in the presence of mono-, di-, and trivalent cations. On high-curvature structures, hybridization current density increases as positive charge increases, whereas on flat electrodes, the trivalent cations cause aggregation due to electrostatic overscreening, which leads to decreased current density and less sensitive detection