Label-free detection of protein–DNA interactions using electrochemical impedance spectroscopy (original) (raw)
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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.
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.
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.
Biosensors and Bioelectronics, 2008
a b s t r a c t DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R ct that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R ct change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R ct change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R ct upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.
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.
DNA biosensor using fluorescence microscopy and impedance spectroscopy
Sensors and Actuators B-chemical, 2006
Two types of DNA biosensors are presented. Both sensing principles are demonstrated using synthetic oligomer single-stranded DNA (ssDNA) with concentrations in the micromolar range. A first sensor type is based on the detection of fluorescently labeled ssDNA to a complementary probe that is bound to a silicon substrate by a disuccinimidyl terephtalate and aminosilane immobilization procedure. An enhanced fluorescent response is obtained using constructive interference effects in a fused silica layer deposited before immobilization onto the silicon substrate. The selectivity of different DNA probes towards complementary and non-complementary DNA targets is tested. A second type of DNA sensor is based on the impedimetric response of a solution of unlabeled 20-mer ssDNA in de-ionized water. Interdigitated microelectrodes that are 5 m wide and separated by 5 m gaps are microfabricated on glass substrates and the complex impedance of the system in the 100 Hz-100 MHz frequency range is investigated. The proportionality between the measured solution resistance and ssDNA concentration is demonstrated.
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.
Detection of abasic DNA by means of impedance spectroscopy
Electrochemistry Communications, 2018
Abasic sites can occur in DNA for different reasons, and thus the detection of this special molecular structure has turned into the focus of research. Here we have investigated the hybridization of abasic ssDNA to immobilized ssDNA probes by impedance spectroscopy. For this purpose three different abasic 25mer ssDNA (abasic site near the electrode; in the middle and near the solution) are studied in comparison to fully complementary 25mer ssDNA. For all abasic strands the surface binding can be followed concentration dependent via impedance spectroscopy; however, the concentration range and the maximum impedance change are found to be different compared to fullmatch ssDNA. Here, the position of the abasic site within the DNA strand significantly determines the signal behavior, and thus even allows a partial discrimination between the different abasic DNA strands. By investigating the binding in parallel by SPR, only slightly smaller surface concentrations are detected for the abasic strands in comparison to the fullmatch strand. This points to the formation of different DNA structures when abasic sites are contained.
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.