Detection Of DNA Hybridization Research Articles

Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL)) modified pencil graphite electrode (IL-PGEs) was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC). The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV) technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC) or mismatched DNA sequence (MM), or and in the presence of mixture of DNA target: NC (1:1) and DNA target: MM (1:1).

The role of polarization-induced reorientation of DNA strands on organic field-effect transistor-based biosensors sensitivity at high ionic strength

The detection of the intrinsic charge of biochemical molecules is a promising strategy for the fabrication of field-effect transistor (FET)-based sensors for direct, non-destructive detection of several biochemical reactions. Nevertheless, the high ionic concentration of standard environments for biochemical species represents a significant limitation to this sensing strategy. Here, an investigation on the physical mechanisms behind the ability of an organic FET-based sensor to detect DNA hybridization at high ionic strengths is proposed. The capability of the device to correctly detect single-stranded DNA oligonucleotides and their hybridization with a complementary target sequence has been analyzed in detail. In particular, the electrical response in solutions with different ionic strengths was investigated and put in relation with the nano-scale properties of DNA strands employed as receptors. Fluorescence analysis shows that it is possible to electrically modify their orientation and consequently improve the device sensitivity in conditions close to those occurring during in vivo hybridization.

E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM).

Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.

Organic FET-based DNA hybridization sensor with sub-picomolar sensitivity.

In this paper, a novel approach for DNA hybridization detection by means of organic field-effect transistors-based biosensors (bioFETs) is proposed. The reported device, namely Organic Charge-Modulated FET (OCMFET), is conceived for overcoming the main limitations of bioFETs, related to design, materials and measurement conditions. In particular, record performances in terms of sensitivity and selectivity will be reported. Moreover, as innovative features among bioFETs, the detection ability of the OCMFET at relatively high ionic strengths will be carefully discussed, unravelling the interactions between the bioreceptors characteristics at the nano-scale and the device polarization.

A genosensor based on CPE for study the interaction between ketamine as an anesthesia drug with DNA

The electrochemical oxidation of ketamine as an analgesia and anesthesia drug and its interaction with DNA was studied at carbon paste electrode (CPE) using voltammetric techniques. Ketamine showed one irreversible oxidation peak nearly around +1.14V vs. Ag|AgCl|KCl (3M) only in Britton buffer (pH 7.00). The effect of scan rate on the cyclic voltammetric behavior of ketamine was investigated at the CPE and binding constant of ketamine and DNA was also calculated. The binding mode of DNA and ketamine was elucidated by differential pulse voltammetry and UV–vis spectroscopy techniques. Based on these results, interaction between ketamine and single-stranded DNA was of electrostatic mode, while between double-stranded DNA and ketamine was of groove binding. Ketamine showed a special affinity toward guanine bases of DNA. Also, ketamine was employed as an electrochemical indicator for detection of DNA hybridization. The difference between the oxidation peak current of the DNA probe modified CPE in the presence and absence of ketamine (ΔI) was enhanced with increasing ketamine concentration and a detection limit of 1.98nM was evaluated. To further investigate the selectivity of this biosensor, some noncomplementary sequences were used. Finally, the proposed method was successfully used for voltammetric determination of ketamine in real samples.

Design of a high figure of merit subwavelength grating based plasmonic sensor for detection of DNA hybridization

A SPR biosensor based on a metallic subwavelength grating structure is studied for detection of DNA hybridization. Performance and dispersion curve characteristics of the sensor are investigated numerically for variations of the grating geometry parameters by finite element method (FEM) as a numerical method. The results of this investigation are used to design a high figure of merit (FOM) SPR sensor in near-infrared (NIR) wavelength of 984nm. Also, performance of the sensor in visible wavelength of 633nm is studied and compared with the NIR one. This study shows that the performance changing with the grating geometry parameters can be elucidated well by evaluating the effects of those parameters especially fill factor (f.f.) on the dispersion curve. Numerical study results in obtaining a higher FOM for operating wavelengths in NIR range in contrary to visible wavelengths because a NIR wavelength can be adjusted as a band edge wavelength locating in the flattened part of the dispersion curve by selecting appropriate values of f.f. and grating thickness.

Mediated Electron Transfer at Vertically Aligned Single-Walled Carbon Nanotube Electrodes During Detection of DNA Hybridization.

Vertically aligned single-walled carbon nanotube (VASWCNT) assemblies are generated on cysteamine and 2-mercaptoethanol (2-ME)-functionalized gold surfaces through amide bond formation between carboxylic groups generated at the end of acid-shortened single-walled carbon nanotubes (SWCNTs) and amine groups present on the gold surfaces. Atomic force microscopy (AFM) imaging confirms the vertical alignment mode of SWCNT attachment through significant changes in surface roughness compared to bare gold surfaces and the lack of any horizontally aligned SWCNTs present. These SWCNT assemblies are further modified with an amine-terminated single-stranded probe-DNA. Subsequent hybridization of the surface-bound probe-DNA in the presence of complementary strands in solution is followed using impedance measurements in the presence of Fe(CN)63−/4− as the redox probe in solution, which show changes in the interfacial electrochemical properties, specifically the charge-transfer resistance, due to hybridization. In addition, hybridization of the probe-DNA is also compared when it is attached directly to the gold surfaces without any intermediary SWCNTs. Contrary to our expectations, impedance measurements show a decrease in charge-transfer resistance with time due to hybridization with 300 nM complementary DNA in solution with the probe-DNA attached to SWCNTs. In contrast, an increase in charge-transfer resistance is observed with time during hybridization when the probe-DNA is attached directly to the gold surfaces. The decrease in charge-transfer resistance during hybridization in the presence of VASWCNTs indicates an enhancement in the electron transfer process of the redox probe at the VASWCNT-modified electrode. The results suggest that VASWCNTs are acting as mediators of electron transfer, which facilitate the charge transfer of the redox probe at the electrode–solution interface.

The portable fluorescence detection system matched with PDMS microfluidic biochip for DNA hybridization detection

The self-made fluorescence detection system was integrated to detect the biological signal of DNA sample. And the portable fluorescence detection system with four channels microfluidic biochip was finished for DNA hybridization.In the biochip preparation, the PDMS cast-forming process was used to fabricate microfluidic biochip with width 150μm and height 200μm. In the fluorescence detection system, four sets of independent 532nm green lasers were adopted to illuminate the four detection areas of the microfluidic biochip for fluorescence sample excitation, then the voltage of excitation fluorescence signal was obtained. Then the PSOC embedded system was utilized synchronously to display the four voltage values on LCD, in the meantime, the voltage value was transferred to the computer for recording.From the experimental result of DNA hybridization, it can be seen that portable fluorescence detection system can be utilized to measure four fluorescence signals of DNA samples at the same time. Moreover, due to the hybridization specificity of fluorescence probe ET996-Hex, the DNA hybridization of Edwardsiella tarda BCRC 16711 and Edwardsiella tarda BCRC 16702 can be judged successfully for fluorescence probe ET996-Hex. The portable fluorescence detection system had the capability to distinguish disease source.

Gold Nanoparticles - An Enhanced DNA Sensing Tools Using Surface Enhance Raman Scattering

Molecular recognition of specific DNA target is crucial for species authentication, drug discovery, forensic investigation and biodiagnostics. Development of nanoparticles at atomic scale and molecular level offers remarkable biocompatibility in the detection of DNA hybridization. Oligo-functionalized nanoparticles with controlled sizes and shapes have come forward with advantages over the previously documented DNA detection assays. Nanomaterials facilitate signal transduction and synergism in biomolecular interaction, promoting both conventional and newly developed DNA identification platforms. Tailoring of DNA sensing tools with gold nanoparticles (Au NPs) enhances the Surface Enhanced Raman Scattering (SERS) signal, allowing target detection with molecular precision. Here we briefly reviewed the recent AuNPs based SERS-platforms for the specific, sensitive and reliable detection of DNA targets for species authentication in foods and feeds.

Au decorated ZnO thin film: application to DNA sensing

Here we reported the gold nanoparticles (GNPs) decorated ZnO thin films based biosensor for the detection of pork DNA hybridization. Label-free DNA detection is increasingly important in bio diagnostics, food analysis and forensic investigations. The capacitance, permittivity and impedance profiles of the biosensor clearly differentiated the complementary and non-complementary target DNA detection using low-cost dielectric analyzer. In this study, GNPs were used to create an attachment surface between the ZnO thin films and the thiol-modified probe DNA and to enhance the sensitivity of the biosensor up to ~59 %. The ZnO thin films and 35 ± 5 nm in diameters of GNP-coating on their surfaces were confirmed with various types of measurement such as FESEM, XRD, EDX and UV---Vis spectroscopy.

Optimization of a horizontal slot waveguide biosensor to detect DNA hybridization.

A full-vectorial H-field formulation-based finite element approach is used to optimize a biosensor structure incorporating a horizontal slot waveguide. It is designed to detect DNA hybridization through the change of the effective index of the waveguide structure. The key parameters, such as normalized power confinement, power density, change in effective index, and sensitivity are presented by optimizing the device parameters of the slot waveguide. It is shown here that a 90.0μm long compact Mach-Zehnder section can be designed with horizontal slot waveguide to detect DNA hybridization and for a ring resonator arrangement a sensitivity of 893.5nm/RIU is obtained.

Detection of DNA Hybridization by Methylene Blue Electrochemistry at Activated Nanoelectrode Ensembles.

Nanoelectrode ensembles (NEEs) obtained by electroless gold deposition in track-etched poly-carbonate (PC) membranes are functionalized and applied for DNA hybridization detection, using methylene blue (MB) as electroactive probe. To this aim, an amine terminated (ss)DNA probe is immobilized on the PC surface of the NEE by reaction via carbodiimide and N-hydroxysulfosuccinimide. In order to increase the number of carboxylic groups present on PC and suitable for the functionalization, the surface of NEEs is oxidized with potassium permanganate. The presence of carboxylic functionalities is verified by spectrochemical titration with thionin acetate (THA) and the effect of the activation treatment on the electrode performances is evaluated by cyclic voltammetry (CV). After activation and functionalization with the probes, the NEE-based sensor is hybridized with complementary target sequences. The effect of the functionalization of the NEEs both with the (ss)DNA probe alone and after hybridization with the target, is studied by measuring the changes in the MB reduction signal by square wave voltammetry (SWV), after incubation in a suitable MB solution, rinsing and transfer to the measurement cell. It was observed that this peak signal decreases significantly after hybridization of the probe with the complementary target. Experimental evidences suggest that the interaction between MB and the guanines of (ss)DNA and (ds)DNA is at the basis of the development of the here observed analytical signal. The proposed approach allows the easy preparation and testing of NEE-based sensors for the electrochemical DNA hybridization detection.

Novel multifunction-integrated molecular beacon for the amplification detection of DNA hybridization based on primer/template-free isothermal polymerization.

Molecular beacon (MB) is widely explored as a signaling probe in powerful biosensing systems, for example, enzyme-assisted strand displacement amplification (SDA)-based system. The existing polymerization-based amplification system is often composed of recognition element, primer, template and fluorescence reporter. To develop a new MB sensing system and simply the signal amplification design, we herein attempted to propose a multifunctional integrated MB (MI-MB) for the polymerization amplification detection of target DNA via introducing a G-rich fragment into the loop of MB without using any exogenous auxiliary oligonucleotide probe. Utilizing only one MI-MB probe, the p53 target gene could trigger the cycles of hybridization/polymerization/displacement, resulting in amplification of the target hybridization event. Thus, the p53 gene can be detected down to 5 × 10(-10)M with the linear response range from 5 × 10(-10)M to 4 × 10(-7)M. Using the MI-MB, we could readily discriminate the point mutation-contained p53 from the wild-type one. As a proof-of-concept study, owing to its simplicity and multifunction, including recognition, replication, amplification and signaling, the MI-MB exhibits the great potential for the development of different biosensors for various biomedical applications, especially, for early cancer diagnosis.

Surface layer reflective index changes of Au nanoparticle functionalized porous silicon microcavity for DNA detection

A technique is demonstrated to detect DNA hybridization based on surface layer of Au/porous silicon microcavity (Au/PSM) substrate for very small amount of biomolecules. Simulations show that the increase of effective refractive index for the first layer of PSM will cause a blue shift for its reflectance spectrum, and the blue shift becomes less with the increase of refractive index for one more layers. In experiments, such a blue shift of reflectance spectrum of PSM comes from the increase of refractive index by DNA hybridization on the surface. The detection limit of Au/PSM biosensor is 15.15 nM for 19-base pair DNA, which is comparable to that of reported biosensors based on porous silicon (PS). Therefore such an Au/PSM could be very useful to develop simple, rapid and sensitive optical biosensors when the amount of target is very small.

Detection of DNA Hybridization Using Protein A Modified Ion Sensitive Field Effect Transistor

A DNA sensor based on an ion sensitive field effect transistor (ISFET) with protein A modification for characterization of DNA hybridization is reported. The surface of the ISFET was modified with protein A via physical adsorption. Protein A binds specifically to the crystallizable fragment of the antibody leading to uniform orientation of the antigen binding site. In order to characterize DNA hybridization, the antibiotin antibody was immobilized to protein A and a single-strand biotinylated DNA probe was added to bind to a specific antibiotin antibody. The voltage shift of the DNA hybridization was observed after the complementary DNA target was added to the immobilized probe. The noncomplementary DNA target was also tested as a negative control. The optimal concentrations were 1 mg/mL for protein A, 1 mg/mL for the anti-biotin antibody, 0.5 µM for the biotinylated probe, and 0.5 µM for the synthetic DNA target. Nevertheless, high concentrations of protein A and antibiotin may prevent the hybridization signal detection; therefore, 0.1 mg/mL of protein A and antibiotin were employed instead of their optimal concentrations. By this strategy, DNA hybridization of the synthetic target was successfully measured with a limit of detection of approximately 0.08 µM. In addition, complementary DNA was differentiated from a noncomplementary DNA target.

Simple and label-free detection of DNA hybridization on a modified graphene nanosheets electrode

In this study, an effective method was devised to synthesize amelogenin genes in solution and to amplify electrical detection of DNA hybridization based on graphene nanosheets (GNs) modified glassy carbon electrode (GCE). GNs are well known as effective biocompatible and conductive materials that can provide large surface area and a sufficient numbers of binding points for DNA immobilization. The biosensor fabrication processes and the electrochemical responses of probe immobilization and hybridization with target DNA were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) using [Fe(CN)6]3−/4− as an electrochemical redox. Due to minimum nonspecific DNA adsorption, a very high specificity of DNA hybridization was achieved, and the hybridization rate of the target DNA in optimum conditions was increased significantly. With this approach, the target DNA could be quantified in a linear range from 1.0×10−20 to 1.0×10−14molL−1 for the first segment and from 1.0×10−13 to 1.0×10−6molL−1 for the second segment, with a detection limit of 7.1×10−21molL−1 by 3sb. In addition, the biosensor exhibited a high level of stability and repeatability, even for the determination of DNA sequences in real samples without amplification.

A nanostructured genosensor for the early diagnosis of systemic arterial hypertension.

The rapid progress of nanomedicine, especially in areas related to medical imaging and diagnostics, has motivated the development of new nanomaterials that can be combined with biological materials for specific medical applications. One such area of research involves the detection of specific DNA sequences for the early diagnosis of genetic diseases, using nanoparticles-containing genosensors. Typical genosensors devices are based on the use of sensing electrodes - biorecognition platforms - containing immobilized capture DNA probes capable of hybridizing with specific target DNA sequences. In this paper we show that upon an appropriate design of the biorecognition platform, efficient sandwich-type genosensors based upon DNA-AuNPs nanocomplexes can be efficiently applied to the detection of a Systemic Arterial Hypertension (SAH) polymorphism located in intron 16 of the Angiotensin-converter enzyme (ACE) gene. Since SAH is intimately related to heart diseases, especially blood hypertension, its early detection is of great biomedical interest. The biorecognition platforms were assembled using mixed self-assembled monolayers (SAMmix), which provided the immobilization of organized architectures with molecular control. Detection of the DNA target sequence at concentrations down to 1 nM was carried out using electrochemical impedance spectroscopy (EIS). We show that the use of EIS combined with specific nanobiocomplexes represents an efficient method for the unambiguous detection of complementary DNA hybridization for preventative nanomedicine applications.

Silicon nanonets for biological sensing applications with enhanced optical detection ability

Optical sensors based on fluorescence methods are used in numerous areas of society, ranging from healthcare to environmental monitoring. But the race to elaborate portable and highly sensitive detection systems leads to the huge development of nanomaterial-based sensors. Here, we have fabricated a silicon nanonet, or silicon nanowire (SiNW) network, ‐based biosensor for DNA hybridization detection by fluorescence microscopy. We demonstrate that by leveraging the properties of the SiNWs such as their large specific surface and the high aspect ratio, these nanonet sensors have significantly enhanced sensitivity and better selectivity compared to plane substrates. The fluorescence signal shows an intensity increasing with the SiNW density on the nanonet and for the denser nanonets, the detection limit for DNA hybridization is 1nM. The elaborated Si nanonet-based DNA sensors present more than 50% change in fluorescence intensity between complementary DNA and 1 base mismatch DNA which shows their high selectivity. Finally, we have integrated the Si nanonet-based sensor into a DNA chip and we have shown that this selective sensor can be reproduced on a large scale area.

Label-Free Detection of DNA Hybridization by Using Charge Perturbation on Poly(thionine)-Modified Glassy Carbon and Gold Electrodes

Simple and label-free electrochemical sensors for the detection of DNA hybridization were developed based on charge perturbation of poly(thionine)-modified glassy carbon and gold electrodes (PTH/GCE and PTH/Au). Probe DNA (pDNA) was immobilized on PTH films via covalent bonds between the amine (-NH2) group of the PTH and the phosphate (PO43−) group at the 5′ end of the pDNA. The immobilized single-strand pDNA decreased the oxidation current of [Fe(CN)6]4− as a result of electrostatic repulsion by the negatively charged PO43- backbone of the pDNA. The current was further decreased subsequent to duplex formation by the hybridization of target complementary DNA (cDNA) due to increased electrostatic repulsion. Both sensors demonstrated excellent selectivity, stability, and the sensitivity of 1.27 and 58.6 μA·cm−2·pM−1 with detection limits of 0.47 and 0.20 pM, respectively, for cDNA hybridization.

Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization.

Rapid detection of disease biomarkers at the patient point-of-care is essential to timely and effective treatment. The research described herein focuses on the development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) DNA aptasensor capable of direct detection of tuberculosis (TB) DNA. Specifically, a plausible DNA biomarker present in TB patient urine was chosen as the model target for detection. Cost-effective screen printed electrodes (SPEs) modified with silver nanoparticles (AgNP) were used as the aptasensor platform, onto which the aptamer specific for the target DNA was immobilized. Direct detection of the target DNA was demonstrated through the appearance of SERS peaks characteristic for adenine, present only in the target strand. Modulation of the applied potential allowed for a sizeable increase in the observed SERS response and the use of thiol back-filling prevented non-specific adsorption of non-target DNA. To our knowledge, this work represents the first EC-SERS study of an aptasensor for the direct, label-free detection of DNA hybridization. Such a technology paves the way for rapid detection of disease biomarkers at the patient point-of-care.