Frequency dependent and surface characterization of DNA immobilization and hybridization (original) (raw)
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Electrical frequency dependent characterization of DNA hybridization
Biosensors and Bioelectronics, 2003
The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5?-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.
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.
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.
Biosensors and Bioelectronics, 1999
A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 g/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.
Small, 2009
In this work, a simple but sensitive electrochemical DNA biosensor for nucleic acid detection was developed by taking advantage of exonuclease (Exo) I-assisted cleavage for background reduction and zirconia-reduced graphene oxide-thionine (ZrO 2-rGO-Thi) nanocomposite for integral DNA recognition, signal amplification, and reporting. The ZrO 2-rGO nanocomposite was obtained by a one-step hydrothermal synthesis method. Then, thionine was adsorbed onto the rGO surface, via π-π stacking, as an excellent electrochemical probe. The biosensor fabrication is very simple, with probe DNA immobilization and hybridization recognition with the target nucleic acid. Then, the ZrO 2-rGO-Thi nanocomposite was captured onto an electrode via the multicoordinative interaction of ZrO 2 with the phosphate group on the DNA skeleton. The adsorbed abundant thionine molecules onto the ZrO 2-rGO nanocomposite facilitated an amplified electrochemical response related with the target DNA. Since upon the interaction of the ZrO 2-rGO-Thi nanocomposite with the probe DNA an immobilized electrode may also occur, an Exo I-assisted cleavage was combined to remove the unhybridized probe DNA for background reduction. With the current proposed strategy, the target DNA related with P53 gene could be sensitively assayed, with a wide linear detection range from 100 fM to 10 nM and an attractive low detection limit of 24 fM. Also, the developed DNA biosensor could differentiate the mismatched targets from complementary target DNA. Therefore, it offers a simple but effective biosensor fabrication strategy and is anticipated to show potential for applications in bioanalysis and medical diagnosis.
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.
DNA Biosensors and Microarrays
Chemical Reviews, 2008
and currently holds a position as a Ph.D. student in biochemistry at the Institut de Chimie et Biochimie Moléculaires et Supramoléculaires at the Université Lyon 1. Her research work focuses on optical biosensors based on polyluminol. Béatrice D. Leca-Bouvier received the Doctorat de Spécialité in biochemistry from the Université Lyon 1 in 1995. After a postdoctoral period at the University of Perpignan, where she worked in Pr. Jean-Louis Marty's group on elecrochemical biosensors, she became Assistant Professor of Biochemistry at the Université Lyon 1, and she is now working on optical biosensors in the Laboratoire de Génie Enzymatique et Biomoléculaire, within the Institut de Chimie et Biochimie Moléculaires et Supramoléculaires. Loïc J. Blum, born in 1955, received the Doctorat de Spécialité (1983) in biochemistry and the Doctorat d'Etat ès Sciences (1991) from the Université Lyon 1. He is presently Professor of Biochemistry and Biotechnology at the same university and is involved in the development of nanobiotechnology-related topics (biosensors, bioanalytical micro-and nanosystems, biochips, and biomimetic membranes). He is the head of both the