Electrochemical detection of DNA hybridization via bis-intercalation of a naphthylimide-functionalized viologen dimer

The synthesis and DNA binding properties of a bis-naphthyl imide tetracationic diviologen compound NI(CH2)3V(2+)(CH2)6V(2+)(CH2)3NI (where V(2+) = 4,4'-bipyridinium and NI = naphthyl imide, NIV) are described. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for reduction of the V(2+) state to the V+ (viologen radical cation) state. Isotherms and binding constants for this molecule to both forms of immobilized DNA were generated in this fashion. The character of the binding isotherm for dsDNA suggests bis-intercalation. Under high saline conditions, the diviologen molecule dissociated 160 times slower from dsDNA compared to ssDNA. Slow dissociation kinetics from dsDNA (kd =7.0 x 10-5 s(-1)) allow this molecule to be used as an effective DNA hybridization indicator.     

Electrochemical imaging of localized sandwich DNA hybridization using scanning electrochemical microscopy

Imaging of localized hybridization of nucleic acids immobilized on gold-DNA chip was performed by means of the feedback mode of scanning electrochemical microscopy (SECM). Thiol-tethered oligodeoxynucleotide (HS-ODN) probes, spotted on a gold surface, were hybridized with unmodified target sequence via sandwich hybridization with a biotinylated signaling probe. Spots where sequence-specific hybridization had occurred were developed by adding a streptavidin-alkaline phosphatase conjugate and biocatalyzed precipitation of an insoluble and insulating product. As a consequence, the surface conductivity of the spotted region of the chip where hybridization had taken place changed. These changes in conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a DNA array in a straightforward way. Analysis of real samples was also performed coupling this method with polymerase chain reaction. The imaging of 60 nM PCR amplicon (255 bp) was demonstrated.     

Electrochemical detection of DNA hybridization by means of osmium tetroxide complexes and protective oligonucleotides

We have utilized protective oligonucleotides to modify DNA fragments with osmium tetroxide complexes without compromising their ability to hybridize with immobilized thiol-linked probe-SAMs on gold electrodes. Due to reversible voltammetric signals of Os(VI/IV), this method allowed sensitive electrochemical hybridization detection of short (25 bases) and long (120 bases) thymine-containing DNA targets. The detection limit was 3.2 nM for the long target. We found an optimum 40 degrees C hybridization temperature for the short target. No interference by noncomplementary DNA was observed. At least 10 repetitive hybridization experiments at the same probe-SAM were possible with thermal denaturation in between. Such use of protective strands could be useful also for other types of DNA recognition and even for other DNA-modifying agents. Moreover, it is possible to produce electrochemically active oligonucleotides (targets and reporter probes) in ones own laboratory in a simple way.     

High-sensitivity detection of DNA hybridization on microarrays using resonance light scattering

The application of resonance light scattering (RLS) particles for high-sensitivity detection of DNA hybridization on cDNA microarrays is demonstrated. Arrays composed of approximately 2000 human genes ("targets") were hybridized with colabeled (Cy3 and biotin) human lung cDNA probes at concentrations ranging from 8.3 ng/microL to 16.7 pg/microL. After hybridization, the arrays were imaged using a fluorescence scanner. The arrays were then treated with 80-nm-diameter gold RLS Particles coated with anti-biotin antibodies and imaged in a white light, CCD-based imaging system. At low probe concentrations, significantly more genes were detected by RLS compared to labeling by Cy3. For example, for hybridizations with a probe concentration of 83.3 pg/microL, approximately 1150 positive genes were detected using RLS compared to approximately 110 positive genes detected with Cy3. In a differential gene expression experiment using human lung and leukemia RNA samples, similar differential expression profiles were obtained for labeling by RLS and fluorescence technologies. The use of RLS Particles is particularly attractive for detection and identification of low-abundance mRNAs and for those applications in which the amount of sample is limited.     

Femtomolar electrochemical detection of DNA hybridization using hollow polyelectrolyte shells bearing silver nanoparticles

The preparation, and use as electrochemical labels, of polyelectrolyte shells bearing Ag nanoparticles is described. Their potential for highly sensitive detection is demonstrated. The shells are prepared by layer-by-layer self-assembly around templates (500 nm diameter) which are then dissolved. The shells can be opened and closed by adjustment of solution pH, and this process is utilized to encapsulate Ag nanoparticles, chiefly by adsorption to the inner walls of the capsules. Based on absorbance, TEM and voltammetric measurements, the highest loading achieved is approximately 78 Ag particles per capsule. The Ag capsules are used via biotin-avidin binding as labels for the detection of DNA hybridization, following acid dissolution and then detection of the Ag (+) by ASV. A 30-mer sequence specific to Escherichia coli is measured at DNA-modified screen-printed electrodes with a detection limit of approximately 25 fM, which corresponds to the detection of 4.6 fg ( approximately 3 x 10 (5) molecules) in the 20 microL analyte sample. A 200 fM target containing a single mismatch gives a significantly (<74%) lower response than 200 fM of complementary target; 60 pM of noncomplementary target gives a negligible response.     

Electrochemical sensing of DNA hybridization based on duplex-specific charge compensation

A nonlabeling voltammetric detection method for DNA hybridization has been developed, in which [Fe(CN)(6)](3-) in solution can readily approach an electrode surface covered with a charge-compensated DNA duplex layer and thus provides a strong redox-sensing current. Charge compensation for negative charges on the DNA backbone has been specifically accomplished on DNA duplexes by discouraging nonspecific binding of positively charged intercalating molecules with single strands. A pretreatment of DNA-modified electrodes with sodium dodecyl sulfate before the intercalator binding process is essential in preventing the nonspecific binding. Since ferricyanide, the only electrochemically active species, is present in the voltammetric solution, the detection signal can be amplified by increasing its concentration. Combination of the duplex-specific charge compensation with the signal amplification has achieved a remarkable signal difference: in 30 mM [Fe(CN)(6)](3-), the area ratio between cyclic voltammograms of the hybridized and unhybridized electrodes is approximately 200 when 3,6-diaminoacridine is used as the intercalator. High sensitivity of the method has been demonstrated by detecting 10 fM (100 zmol in amount) of a target probe DNA.     

Chip and solution detection of DNA hybridization using a luminescent zwitterionic polythiophene derivative

Electronic polymers in aqueous media may offer bioelectronic detection of biospecific interactions. Here we report a fluorometric DNA hybridization detection method based on non-covalent coupling of DNA to a water-soluble zwitterionic polythiophene derivative. Introduction of a single-stranded oligonucleotide will induce a planar polymer and aggregation of the polymer chains, detected as a decrease of the intensity and a red-shift of the fluorescence. On addition of a complementary oligonucleotide, the intensity of the emitted light is increased and blue-shifted. The detection limit of this method is at present approximately 10(-11) moles. The method is highly sequence specific, and a single-nucleotide mismatch can be detected within five minutes without using any denaturation steps. The interaction with DNA and the optical phenomena persists when the polymer is deposited and patterned on a surface. This offers a novel way to create DNA chips without using covalent attachment of the receptor or labelling of the analyte.     

Direct detection of DNA conformation in hybridization processes

DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays.     

DNA hybridization using surface plasmon-coupled emission

We describe a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). Excited fluorophores are known to couple with surface oscillations of electrons in thin metal films, typically 50 nm thick silver on a glass prism. These surface plasmons then radiate into the glass at a sharply defined angle determined by the emission wavelength and the optical properties of the glass and metal. This radiation has the same spectral profile as the emission spectrum of the fluorophores. We studied the emission due to Cy3-labeled DNA oligomers bound to complementary unlabeled oligomers which were themselves bound to the metal surface. Hybridization resulted in SPCE due to Cy3-DNA into the prism. Directional SPCE was observed whether the sample was illuminated from the sample side or through the glass substrate at the surface plasmon angle for the excitation wavelength. A large fraction of the total potential emission is coupled to the surface plasmons resulting in improved sensitivity. When illuminated through the prism at the surface plasmon angle, the sensitivity is increased due to the enhanced intensity of the resonance evanescent field. It is known that SPCE depends on proximity to the silver surface. As a result, changes in emission intensity are observed due to fluorophore localization even if hybridization does not affect the quantum yield of the fluorophore. The use of SPCE resulted in suppression of interfering emission from a noncomplementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. We expect SPCE to have numerous applications to nucleic acid analysis and for the measurement of bioaffinity reactions.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.     

Indium microrod tags for electrochemical detection of DNA hybridization

The preparation and advantages of indium microrod tracers for solid-state electrochemical detection of DNA hybridization are described. The cylindrical metal particles were prepared by a template-directed electrochemical synthetic route involving plating of indium into the pores of a host membrane. The linear relationship between the charge passed during the preparation and the resulting particle size allows tailoring of the sensitivity of the electrical DNA assay. The resulting micrometer-long rods thus offer a greatly lower detection limit (250 zmol), as compared to common bioassays' spherical nanoparticle tags. Indium offers a very attractive electrochemical stripping behavior and is not normally present in biological samples or reagents. Solid-state derivative-chronopotentiometric measurements of the indium tracer have been realized through a "magnetic" collection of the DNA-linked particle assembly onto a thick-film electrode transducer. Factors affecting the performance, including the preparation of the microrods and pretreatment of the transducer surface, were evaluated and optimized. The resulting protocol offers great promise for other affinity bioassays, as well as for electrical coding and identification (through the plating of different metal markers and of multimetal redox-encoded tags).     

Electrochemical detection of glutathione using redox indicators

The nucleophilic addition of the aminothiols homocysteine (HCY), cysteine (CYS), and glutathione (GSH) to the electrogenerated quinone of fluorone black (1) via the ECE mechanism is reported. It is demonstrated that 1selectively reacts with GSH to form the bis-GSH adduct, 1-(GSH)(2), while only the monothiol adducts were generated in the presence of HCY and CYS (1-HCY and 1-CYS, respectively). The more anodic E(pa) of 1-(GSH)2 relative to 1 and 1-GSH (DeltaE(pa) approximately +0.14) is the voltammetric signature that allows the discrimination of GSH from HCY and CYS. It is also shown that the presence of structurally similar aminothiols-HCY and CYS-posed no interference to the signature voltammetric response of 1-(GSH)2.     

Metal nanoparticle-based electrochemical stripping potentiometric detection of DNA hybridization

A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to the bridging of the gold nanoparticles to the magnetic beads. Further signal amplification, and lowering of the detection limits to the nanomolar and picomolar domains, are achieved by precipitating gold or silver, respectively, onto the colloidal gold label. The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.     

Multiplexed hybridization detection of quantum dot-conjugated DNA sequences using surface plasmon enhanced fluorescence microscopy and spectrometry

In this study, the general suitability of quantum dot (QD)-DNA conjugates for the surface plasmon enhanced fluorescence spectroscopy technique is demonstrated. Furthermore, the QD-DNA system is transferred to the platform of surface plasmon enhanced fluorescence microscopy. Using this technique together with a microarray format, in which the sensor-bound single-stranded catcher probes are organized in individual surface spots, results in a simultaneous qualitative analysis of QD-conjugated analyte DNA strands as multicolor images. A clear decomposition of different QD(x)()-DNA(y)() mixtures can be achieved for sequential, as well as mixture injections. Besides this, the study describes the successful approach of measuring spectrally resolved surface plasmon enhanced fluorescence signals derived from catcher probe hybridized QD-DNA conjugates.     

Magnetic nanoparticles applied in electrochemical detection of controllable DNA hybridization

Electrochemical detection of hybridized DNA strands was achieved with a magnetic nanoparticle modified electrode and the commonly used electrochemical couple K3[Fe(CN)6]/K4[Fe(CN)6]. The detection proved to be fast and very simple. Furthermore, magnetic nanoparticles could be employed to control the DNA hybridization process. An inhibited or an enhanced degree of hybridizing could be produced.     

Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).     

Direct DNA hybridization detection based on the oligonucleotide-functionalized conductive polymer

Electrochemical methods for DNA hybridization detection have many advantages that are very fast to detect hybridization and can be directly applied for a portable DNA sensor. In this paper, an electrochemical method to directly detect DNA hybridization was developed on the basis of a new conductive polymer, which was polymerized on the glassy carbon electrode with a terthiophene monomer having a carboxyl group (3'-carboxyl-5,2',5',2"-terthiophene). The ss-DNA probe was made by chemically bonding an amine-linked C6 alkyl group to the 5' terminus of oligonucleotide (19-mer). The probe moiety was immobilized on the polymer through covalent bonding with a catalyst, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. A difference in admittance was observed before and after hybridization as a result of the reduction of the resistance after hybridization. The highest difference in admittance was observed around 1 kHz before and after hybridization. Hybridization amounts of end two-base and center one-base mismatched sequences were obtained only in a 14.3% response when compared to that for the complementary matched sequence.     

Chip-based optical detection of DNA hybridization by means of nanobead labeling

A new scheme for the detection of molecular interactions based on optical readout of nanoparticle labels has been developed. Capture DNA probes were arrayed on a glass chip and incubated with nanoparticle-labeled target DNA probes, containing a complementary sequence. Binding events were monitored by optical means, using reflected and transmitted light for the detection of surface-bound nanoparticles. Control experiments exclude significant influence of nonspecific binding on the observed contrast. Scanning force microscopy revealed the distribution of nanoparticles on the chip surface.     

Label-free detection of DNA hybridization using pyrene-functionalized single-walled carbon nanotubes: effect of chemical structures of pyrene molecules on DNA sensing performance

We investigate the effect of functional groups of pyrene molecules on the electrical sensing performance of single-walled carbon nanotubes (SWNTs) based DNA biosensor, in which pyrenes with three different functional groups of carboxylic acid (Py-COOH), aldehyde (Py-CHO) and amine (Py-NH2) are used as linker molecules to immobilize DNA on the SWNT films. UV/Visible absorption spectra results show that all of the pyrene molecules are successfully immobilized on the SWNT surface via pi-pi stacking interaction. Based on fluorescence analysis, we show that the amide bonding of amine terminated DNA via pyrene containing carboxylic groups is the most efficient to immobilize DNA on the nanotube film. The electrical detection results show that the conductance of Py-COOH modified SWNT film is increased upon DNA immobilization, followed by further increase after hybridization of target DNAs. It indicates that the pyrene molecules with carboxylic acid groups play an important role to achieve highly efficient label-free detection by nondestructive and specific immobilization of DNAs.