Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes (original) (raw)
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Biosensors and Bioelectronics, 2008
The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) × 10 12 /cm 2 . The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 × 10 12 /cm 2 , electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.
A label-free DNA sensor based on impedance spectroscopy
Electrochimica Acta, 2008
This paper describes a label-free detection system for DNA strands based on gold electrodes and impedance measurements. A single-stranded 18 mer oligonucleotide (ssDNA) was immobilised via a thiol linker on gold film electrodes and served as probe DNA. Residual binding places were filled with mercaptobutanol. The sensor surface clearly distinguished between complementary and non-complementary target ssDNA. Additionally, detection of single base pair mismatches was possible. The electrode was impedimetrically characterised in the presence of the redox system ferri/ferrocyanide before and after DNA hybridisation. Impedance analysis showed that the charge transfer resistance, R ct , was increasing after DNA duplex formation, whereas the capacitive properties remain rather unaltered. The relative change of R ct was used as sensor parameter. Concentrations in the nanomolar range have been detected by the system. The sensor was reusable because a denaturation protocol allowed effective double strand dissociation without changing the surface properties of the electrode substantially. The time for DNA detection have been reduced to about 15 min including regeneration. The sensor signal was amplified by about 20% after binding of a negatively charged molecule to the formed DNA duplex. The sensor was also capable of sensing longer target ssDNA strands as shown with 25 mer and 37 mer oligonucleotides.
Label-free detection of protein–DNA interactions using electrochemical impedance spectroscopy
Electrochimica Acta, 2011
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.
In pathogen diagnosis, a single stranded target DNA may be detected in a sensor carrying an active surface with a recognition layer of its complementary single stranded DNA (ssDNA probe). Such a surface may be the core of a specific DNA sensing electrode. The density of probe ssDNA should then be optimized to display the greatest sensitivity to the target DNA. In this work we utilize the electrochemical impedance spectroscopy (EIS) on the electrode reactions for ferro/ferricyanide ([Fe(CN) 6 ] 4-/3-) and methylene blue (MB) to characterize different stages along the development of an indium tin oxide (ITO)-DNA sensor for target ssDNA after PCR amplification. In particular, we study the effects of applied DC potential pretreatments on the ferro/ferricyanide-electrode interaction caused. The effect of the electrostatic repulsion by negative charges of single stranded DNA (ssDNA) was observed to affect the rate of the overall reaction rates for the redox couples. On one hand, EIS spectra for the [Fe(CN) 6 ] 4-/3reaction on DNA modified ITO surfaces yielded time-independent fitting parameters which were consistent with the scanning electrochemical microscopy (SECM) images. On the other hand, MB species yielded time-dependent parameters due to adsorption and intercalation processes that take place on DNA. Models were proposed to explain such time-dependent behavior. The results of the impedance measurements were explained in terms of the variation of the surface charge for different densities of probe ssDNA.
Electroanalysis, 2009
Actinomycin D or proflavine which are known to intercalate within the helix of double-stranded DNA (dsDNA) are used as label-free control to unequivocally prove complementary DNA hybridization by means of electrochemical impedance spectroscopy (EIS). Based on a carefully designed interface comprising a thiol-tethered (20mer) oligonucleotide capture probe which forms a self-assembled monolayer on a gold electrode together with a short chain hydroxyl-terminated alkylthiol, formation of dsDNA can be monitored by an increase of the charge-transfer resistance of a free-diffusing negatively charged redox species ([Fe(CN) 6 ] 3À/4À ). The increase of the charge transfer resistance due to complementary hybridization was about 10 times from the unmodified Au surface to the dsDNA modified electrode. Specific interaction of intercalators with dsDNA leads to a decrease in charge transfer resistance due to the conformational changes in the dsDNA monolayer and partial charge compensation caused by the positively charged intercalators. No shift in the charge transfer resistance was observed in case of incubation of a ssDNA surface with intercalators or when hybridization was invoked using a noncomplementary DNA sequence. Thus, hybridization can be unambiguously detected using EIS by first recording the increase in charge-transfer resistance due to hybridization with the matching target strand followed by recording a decrease in charge transfer resistance caused by intercalation. Nonspecific adsorption can hence be doubtlessly excluded as a reason for the observed changes in the impedance spectrum.
An Electrochemical DNA Biosensor Developed on a
2008
An electrochemical DNA nanobiosensor was prepared by immobilization of a 20mer thiolated probe DNA on electro-deposited generation 4 (G4) poly(propyleneimine) dendrimer (PPI) doped with gold nanoparticles (AuNP) as platform, on a glassy carbon electrode (GCE). Field emission scanning electron microscopy results confirmed the co-deposition of PPI (which was linked to the carbon electrode surface by C-N covalent bonds) and AuNP ca 60 nm. Voltammetric interrogations showed that the platform (GCE/PPI-AuNP) was conducting and exhibited reversible electrochemistry (E°′ = 235 mV) in pH 7.2 phosphate buffer saline solution (PBS) due to the PPI component. The redox chemistry of PPI was pH dependent and involves a two electron, one proton process, as interpreted from a 28 mV/pH value obtained from pH studies. The charge transfer resistance (Rct) from the electrochemical impedance spectroscopy (EIS) profiles of GCE/PPI-AuNP monitored with ferro/ferricyanide (Fe(CN)63-/4-) redox probe, decreased by 81% compared to bare GCE. The conductivity (in PBS) and reduced Rct (in Fe(CN)63-/4-) values confirmed PPI-AuNP as a suitable electron transfer mediator platform for voltammetric and impedimetric DNA biosensor. The DNA probe was effectively wired onto the GCE/PPI-AuNP via Au-S linkage and electrostatic interactions. The nanobiosensor responses to target DNA which gave a dynamic linear range of 0.01 - 5 nM in PBS was based on the changes in Rct values using Fe(CN)63-/4- redox probe.
Surface chemistry effects on the performance of an electrochemical DNA sensor
Bioelectrochemistry, 2009
E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.
Langmuir, 1999
A novel method for the sensitive and specific electrochemical analysis of DNA is described using Faradaic impedance spectroscopy. A thiol-thymine-tagged oligonucleotide (1) capable of forming only one doublestranded turn with the target DNA analyte (2) is assembled on a Au electrode and acts as the sensing interface. The resulting functionalized electrode is reacted with a complex between the target DNA (2) and a biotinylated oligonucleotide (3) to yield a bifunctional double-stranded assembly on the electrode support. The Faradaic impedance spectra, using Fe(CN)6 3as redox probe, reveal an increase in the electrontransfer resistance at the electrode surface upon the construction of the double-stranded assembly. This is attributed to the electrostatic repulsion of Fe(CN)6 3upon formation of the negatively charged doublestranded superstructure. Binding of an avidin-HRP conjugate to the oligonucleotide-DNA assembly further insulates the electrode and increases the interfacial electron-transfer resistance. The HRP-mediated biocatalyzed oxidation of 4-chloro-1-naphthol (4) by H2O2 yields a precipitate (5) on the conductive support and stimulates a very high barrier for interfacial electron transfer, Ret ) 14.7 kΩ. Thus, the precipitation of 5 confirms and amplifies the sensing process of the target DNA (2). The analyte DNA (2) corresponds to the mutated gene fragment characteristic of the Tay-Sachs genetic disorder. The normal gene (2a) is easily discriminated by the sensing interface. The sensor device enables detection of the target DNA (2) with a sensitivity of at least 20 × 10 -9 g‚mL -1 . Cyclic voltammetry experiments further confirm the formation of barriers for the interfacial electron transfer upon the buildup of the double-stranded oligonucleotide-DNA structure and upon the biocatalytic deposition of 5 on the electrode surface.