Chip-based electrical detection of DNA

A variety of methods have been developed for the detection of the binding of the complementary strand of DNA to a gene chip using electrical rather than the established optical signal techniques. Chip-based DNA sensors offer sensitivity, specificity, parallelisation and miniaturisation for the detection of selected DNA sequences or mutated genes associated with human diseases. Problems associated with the established fluorescence-based optical detection technique include the high equipment costs and the need to use sophisticated numerical algorithms to interpret the data. These problems generally limit its use to research laboratories and make it hard to adapt this detection scheme for on-site or point-of-care use. An electrical readout might be a solution to these problems. A review of a number of different approaches to achieve an electrical readout for a DNA chip is presented. The review covers various methods that are based on the use of metal nanoparticles as labels and also electrochemical methods that use polymer-modified electrodes, DNA-specific redox reporters, and DNA-mediated charge transport techniques.     

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.     

Chip-based microelectrodes for detection of single-nucleotide mismatch

Microelectrode arrays having eight 10-microm-diameter gold microelectrodes arranged on a gold-covered Si chip were designed and characterized. The chips prove useful for the detection of single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy.     

Ultrasensitive detection of DNA sequences in solution by specific enzymatic labeling

We present a newly developed technique for the direct detection of very low concentrations of specific nucleic acid sequences in homogeneous solution based on a polymerase extension reaction. This method consists of synthesizing a highly fluorescent nucleic acid reporter molecule using a sequence of the target as a template. Synthesis of the reporter molecule is accomplished by hybridizing a short complementary oligonucleotide primer to the target and extending the reporter using a polymerase and free nucleotides. One of these nucleotides is partially labeled with a fluorophore. The reaction sample is then flowed through the capillary cell of a single molecule detector. Detection of the reporter signifies the presence of the target being sought. Under carefully selected conditions, fluorescence from the reporter molecule is much stronger than that of the free nucleotide background over the detection time. We have derived practical equations that allow us to determine an optimal range of values for the relative reporter and free-nucleotide concentrations. This method allows for the rapid, direct detection of individual targets at femtomolar concentrations without the use of an amplification procedure, such as the polymerase chain reaction.     

Simultaneously amplified electrochemical and surface plasmon optical detection of DNA hybridization based on ferrocene-streptavidin conjugates

A sensitive method based on ferrocene-streptavidin (Fc-Stv) conjugates for the simultaneously amplified electrochemical and surface plasmon optical detection of DNA target hybridization to peptide nucleic acid-modified gold surfaces is reported. The attachment of Fc-Stv to the biotinylated complementary target DNA not only amplified the surface plasmon resonance signal but also enhanced the electrochemical signal due to the many Fc markers per Stv. The ferrocene redox peak current increased with the increase of the target DNA concentration. Consequently, the amount of hybridized target DNA can be estimated by cyclic voltammetry and chronocoulometry. The detection limit of this DNA sensor is 10 pM (2 fmol, with signal to noise > 3). This sensor was also shown to have high selectivity (at the single-base mismatch level) and good reproducibility.     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.     

Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization

We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.     

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.     

"Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes

Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.     

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.     

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).     

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.     

Chip-based scanometric detection of mercuric ion using DNA-functionalized gold nanoparticles

We have developed a chip-based scanometric method for the detection of mercuric ion (Hg (2+)). This method takes advantage of the cooperative binding and catalytic properties of DNA-functionalized gold nanoparticles and the selective binding of a thymine-thymine mismatch for Hg (2+). The limit of detection of this assay in buffer and environmentally relevant samples (lake water) is 10 nM (2 ppb) Hg (2+), which is the U.S. Environmental Protection Agency (EPA) limit of [Hg (2+)] for drinkable water and 1 order of magnitude lower than previous colorimetric assays. This assay is capable of discriminating Hg (2+) from 15 other environmentally relevant metal ions. The method is attractive for potential point-of-use applications due to its high throughput, convenient readout, and portability.     

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.     

Label-free detection of DNA hybridization at a liquid|liquid interface

A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of ion-transfer resistance. The detection limit was of 10-8 M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters.     

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.     

Silver nanoparticle-based ultrasensitive chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which the DNA targets are first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells and then the silver nanoparticles modified with alkylthiol-capped oligonucleotides are used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, silver nanoparticles are dissolved to Ag+ in HNO3 solution and sensitively determined by a coupling CL reaction system (Ag+-Mn2+-K2S2O8-H3PO4-luminol). The combination of the remarkable sensitivity of the CL method with the large number of Ag+ released from each hybrid allows the detection of specific sequence DNA targets at levels as low as 5 fM. The sensitivity increases 6 orders of magnitude greater than that of the gold nanoparticle-based colorimetric method and is comparable to that of surface-enhanced Raman spectroscopy, which is one of the most sensitive detection approaches available to the nanoparticle-based detection for DNA hybridization. Moreover, the perfectly complementary DNA targets and the single-base mismatched DNA strands can be evidently differentiated through controlling the temperature, which indicates that the proposed CL assay offers great promise for single-nucleotide polymorphism analysis.     

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.     

多层次分割实现微小尺寸实时测量

针对灰度图像多个斜坡状边缘目标的微小尺寸实时检测,提出了两次阈值分割的检测算法。预分割提取目标并确定其过渡区。而后分割是在过渡区搜索目标真实边缘,实现尺寸测量。实验表明,这种两次阈值分割算法对 0.5mm 以下目标测量优势明显,精度优于 0.001mm,并降低了噪声对真实边缘点数目的影响。在实时性能上,测量平均时间为 60ms,比拟合算法缩短了 40%,保证了检测系统下位机 2000 packages/h 的处理速率。