A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes

A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)₂dppx²⁺) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and sensitivity of the two probes were compared.     

Sensitive sequence-specific molecular identification system comprising an aluminum micro-nanofluidic chip and associated real-time confocal detector

A method for the development of continuous density gradients of immobilized oligonucleotide probes (20mer) along the length of microfluidic channels is demonstrated. The development of continuous density gradients was achieved using variable electrokinetic transport of probes in hybrid glass-polydimethylsiloxane microfluidic chips. The probes were terminated with an amine functional group, and were delivered by electrokinetic pumping to the flat glass channel wall after it had been densely coated with covalently immobilized aldehyde groups. This method provided probe immobilization densities ranging from 4.5(±0.8)×10(13) to 2.5(±0.8)×10(11) molecules cm(-2), with longitudinal dilution and differential mass transport of the injected plug of probes being the primary factors responsible for the gradient of density. The utility of the resulting density gradient of immobilized probes to control the selectivity of hybridization was demonstrated at room temperature by discrimination between a fully complementary oligonucleotide target, and a target strand containing 3 base pair mismatches (3 BPM) based on the spatial pattern of hybridization for sub-picomole quantities of targets. Single nucleotide polymorphism (SNP) discrimination was possible when temperature control was implemented to improve resolution of the mismatch discrimination, allowing SNP discrimination at 35 °C with a contrast ratio of almost 5 to 1.     

Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences

A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH₂-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO₃ and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 × 10⁻¹² to 4.0 × 10⁻⁹ mol L⁻¹ with a detection limit of 2.75 × 10⁻¹² mol L⁻¹ (3σ). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis

In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10⁻¹¹, 2.4 × 10⁻¹¹ and 3.0 × 10⁻¹¹ M, respectively. The multiplex assay also provided good specificity without any cross-reaction.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

DNA Hybridization at Magnetic Nanoparticles with Electrochemical Stripping Detection

A simple and practical method for electrochemical DNA hybridization assay has been developed to take advantage of magnetic nanoparticles for ssDNA immobilization and zinc sulfide nanoparticle as oligonucleotide label. Magnetic nanoparticles were prepared by coprecipitation of Fe²⁺ and Fe³⁺ with NH₄OH, and then amino silane was coated onto the surface of magnetite nanoparticles. The magnetic nanoparticles have the advantages of easy preparation, easy surface modification and low cost. The target ssDNA with the phosphate group at the 5′ end was then covalently immobilized to the amino group of magnetite nanoparticles by forming a phosphoramidate bond in the presence of 1‐ethyl‐3‐(3‐dimeth‐ylaminopropyl)carbodiimide (EDAC). The zinc sulfide (ZnS) nanoparticle‐labeled oligonucleotides probe was used to identify the target ssDNA immobilized on the magnetic nanoparticles based on a specific hybridization reaction. The hybridization events were assessed by the dissolution of the zinc sulfide nanoparticles anchored on the hybrids and the indirect determination of the dissolved zinc ions by anodic stripping voltammetry (ASV) at a mercury film glassy carbon electrode (GCE). The proposed method couples the high sensitivity of anodic stripping analysis for zinc ions with effective magnetic separation for eliminating nonspecific adsorption effects and offers great promise for DNA hybridization analysis.     

Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe

The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA. A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)₃₀. Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment. The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail. This allowed the subsequent determination of all hybrids through the use of the (dA)₃₀-aequorin conjugate as a universal reagent. The bound aequorin was measured by adding Ca²⁺ and integrating the light emission for 3 s. As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2. The analytical range extended up to 2000 pM. As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2. The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5–19.8%. The use of a (dA)₃₀-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

Extended Langmuir approach to analyze the telomeric sequence using a biochromatographic concept

Immobilization of single-stranded DNA (ssDNA) on chromatographic support, i.e. copolymerized particles of styrene and glycidyl methacrylate via formation of amino groups with either N-methyl-1,3-propanediamine (stationary phase 1) or hexamethylenediamine (stationary phase 2) is a promise method to separate sequence specific DNA. However, the low ligand density of nonporous particles led to nonspecific interaction of the complementary oligonucleotide DNA in the sample with the stationary phase (1 or 2). In this paper, the binding process of telomere (tel; TTAGGG) to the complementary ssDNA immobilized (AATCCC) on the two chromatographic supports was studied for the first time using extended Langmuir equation. It appeared that the nonspecific adsorption of the tel due to electrostatic interaction between the polynucleotide sample (tel) and the residual amino groups on the particle surface via amination with hexamethylenediamine (stationary phase 2) was significant and could be reduced by using a high salt (NaCl) concentration in the bulk solvent. In contrast, the nonspecific adsorption of tel was neglected in the column using DNA-immobilized particles via amination with N-methyl-1,3-propanediamine (stationary phase 1). Thus, the affinity chromatography prepared via amination by N-methyl-1,3-propanediamine was more effective for analysis of sequence-specific DNA than the one prepared via amination by hexamethylenediamine.Moreover, for the two stationary phases, increasing NaCl concentration in the bulk solvent enhanced the hybridization between tel and the complementary immobilized oligonucleotide.     

New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces

A new approach to oligonucleotide arrays is demonstrated that utilizes zirconium phosphonate-derivatized glass slides. The active slides are prepared by binding Zr(4+) to surfaces terminated with organophosphonate groups previously deposited using either Langmuir-Blodgett or self-assembled monolayer methods. Oligonucleotide probes modified with a terminal phosphate bind strongly to the active zirconium phosphonate monolayer, and arrays for detecting fluorescent targets have been prepared using commercial spotting and scanning instruments. Preferred binding to the surface of the terminal phosphate of the modified probes instead of the internal phosphate diester groups is demonstrated and shown to yield increased fluorescence intensity after hybridization with labeled targets. A significant decrease in background signal is achieved by treating the slides with bovine serum albumin after spotting and before hybridization. A further increase in fluorescence after hybridization is observed when using a poly-guanine spacer between the probe oligomer and the terminal phosphate. Combining these modifications, an intensity ratio of nearly 1000 is achieved when comparing 5'-phosphate-modified 33-mer probes with unmodified probes upon hybridization with fluorescent targets.     

Computer simulation study of probe-target hybridization in model DNA microarrays: effect of probe surface density and target concentration

We use lattice Monte Carlo simulations to study the thermodynamics of hybridization of single-stranded "target" genes in solution with complementary "probe" DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct, with each segment representing a sequence of nucleotides that interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how surface density (number of probes per unit surface area) and concentration of target molecules affect the extent of hybridization. For short probe lengths, as the surface density increases, the probability of binding long stretches of target segments increases at low surface density, reaches a maximum at an intermediate surface density, and then decreases at high surface density. Furthermore, as the surface density increases, the target is less likely to bind completely to one probe; instead, it binds simultaneously to multiple probes. At short probe lengths, as the target concentration increases, the fraction of targets binding completely to the probes (specificity) decreases. At long probe lengths, varying the target concentration does not affect the specificity. At all target concentrations as the probe length increases, the fraction of target molecules bound to the probes by at least one segment (sensitivity) increases while the fraction of target molecules completely bound to the probes (specificity) decreases. This work provides general guidelines to maximizing microarray sensitivity and specificity. Our results suggest that the sensitivity and specificity can be maximized by using probes 130-180 nucleotides long at a surface density in the range of 7 x 10(-5)- 3 x 10(-4) probe molecules per nm(2).     

Microplate electrochemical DNA detection for phosphinothricin acetyltransferase gene sequence with cadmium sulfide nanoparticles

An electrochemical DNA detection method for the phosphinothricin acetyltransferase (PAT) gene sequence from the transgenetic plants was established by using a microplate hybridization assay with cadmium sulfide (CdS) nanoparticles as oligonucleotides label. The experiment included the following procedures. Firstly target PAT ssDNA sequences were immobilized on the polystyrene microplate by physical adsorption. Then CdS nanoparticle labeled oligonucleotide probes were added into the microplate and the hybridization reaction with target ssDNA sequences took place in the microplate. After washing the microplate for three times, certain amounts of HNO₃ were added into the microplate to dissolve the CdS nanoparticles anchored on the hybrids and a solution containing Cd²⁺ ion was obtained. At last differential pulse anodic stripping voltammetry (DPASV) was used for the sensitive detection of released Cd²⁺ ion. Based on this principle a sensitive electrochemical method for the PAT gene sequences detection was established. The voltammetric currents of Cd²⁺ were in linear range with the target ssDNA concentration from 5.0 × 10^− 13 to 1.0 × 10^− 10 mol/L and the detection limit was estimated to be 8.9 × 10^− 14 mol/L (3σ). The proposed method showed a good promise for the sensitive detection of specific gene sequences with good selectivity for the discrimination of the mismatched sequences.     

DNA detection method based on the two-dimensional aggregation and selective desorption of nanoparticle probes

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.     

Nanostructured zirconium oxide based genosensor for Escherichia coli detection

A deoxyribonucleic acid (DNA) biosensor has been fabricated via immobilization of 17 base terminal single stranded DNA (ssDNA) identified from the 16s rRNA coding region of Escherichia coli onto sol–gel derived nanostructured zirconium oxide (NanoZrO₂) film. An oligonucleotide probe with a terminal 5′-phosphate group has been attached to the surface of the electrode via affinity of NanoZrO₂ for phosphate. The results of hybridization studies carried out with the complementary, non-complementary and genomic DNA reveal that ssDNA/NanoZrO₂/ITO bioelectrode has a high selectivity and sensitivity towards hybridization detection with limits of 10^?6–10⁶ pM of complementary DNA.     

Controlling DNA orientation on mixed ssDNA/OEG SAMs

We present and characterize a mixed self-assembled monolayer (SAM) consisting of single-stranded oligonucleotide (ssDNA)- and oligo(ethylene glycol) (OEG)-terminated thiols. The ssDNA/OEG SAMs are prepared by simultaneous coadsorption from a common thiol solution over a broad range of compositions. Electron spectroscopy for chemical analysis (ESCA) is used to measure the surface coverage of ssDNA, whereas surface plasmon resonance (SPR) sensor is used to measure the hybridization of complementary ssDNA and protein resistance. Through the complementary use of these techniques, we find that the composition of OEG in the assembly solution controls a key parameter: the surface coverage of ssDNA on the surface. There is evidence that it influences the orientation of the immobilized ssDNA probes. Lower OEG concentrations yield a surface with higher ssDNA coverage and less favorable orientation, whereas higher OEG concentrations produce a surface with lower DNA coverage and more favorable orientation. Competition between these two effects controls the hybridization efficiency of the ssDNA surface. Compared to ssDNA surfaces prepared with other diluent thiols, the use of OEG improves the protein resistance of the surface, making it more broadly applicable.