Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT₂₀ as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature (T_m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T_m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.     

Capillary waveguide nucleic acid based biosensor

Micro-capillaries are finding increasing utility in the development of portable analytical sensors. We present design guidelines for optimizing the collection of free propagating fluorescence for capillary waveguide sensors used in the detection of nucleic acids. A dual function integrated opto/fluid connector is also described. Evanescent wave excitation of the coating layer containing a DNA probe is achieved by using a fiber optic ring arrangement for coupling light directly into the capillary wall. The central part of the connector is used for injecting a DNA or RNA target into the capillary channel. In situ hybridization has been used to detect target molecules at a concentration of 30 pg ml⁻¹. The sensor can be regenerated for repeated detection of DNA or RNA targets.     

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.     

Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective ‘reagent’ to bind complementary DNA or RNA for applications such as the detection of pathogenic organisms, gene therapy agents and genetic mutations. The density of ssDNA on a surface will determine the charge density due to ionizable phosphate groups. Such a negatively charged interface will attract positive counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions which influence the thermodynamics of hybridization, and this can studied by the melt temperature (T_m) of double-stranded DNA (dsDNA). Experimental work and theoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and therefore, changes in selectivity and drift in calibration for quantitative analysis. Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT₂₀ that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica that was coated with DNA were used in a total internal reflection fluorescence instrument to determine T_m from the dissociation of duplexes of fluorescein-labeled dA₂₀ : dT₂₀. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA and on the extent of hybridization of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk solution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a reduced dielectric constant within a DNA layer relative to that in bulk solution.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

A microfluidic biosensor based on nucleic acid sequence recognition

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol L^–1 (10 pmol L^–1) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.     

Trends in the development of nucleic acid biosensors for medical diagnostics

Some of the recent advances in the field of biosensors for nucleic acid analysis in medical diagnostic applications are highlighted. Particular attention is paid in this review to the progress made in two key areas of development: (i) enhancements achieved in device selectivity, and (ii) enhancements achieved in device sensitivity.     

Surface plasmon resonance sensing of nucleic acids: A review

Biosensors based on surface plasmon resonance (SPR) have become a central tool for the investigation and quantification of biomolecules and their interactions. Nucleic acids (NAs) play a vital role in numerous biological processes and therefore have been one of the major groups of biomolecules targeted by the SPR biosensors. This paper discusses the advances of NA SPR biosensor technology and reviews its applications both in the research of molecular interactions involving NAs (NA–NA, NA–protein, NA–small molecule), as well as for the field of bioanalytics in the areas of food safety, medical diagnosis and environmental monitoring.     

A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid

A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3′-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully.     

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

A novel detection of radon based on its decay product inducing conformational changes of an aptamer probe

This study proposes a novel method for the detection of inert gas radon using a label-free, specific, fluorescence-sensing aptamer in the context of PW17-OG system. This method utilizes the cyanine dye OliGreen (OG) as a signal reactor and the aptamer PW17 as a fluorescent identification probe. When OG integrates into the free curling PW17, a strong fluorescence signal is generated. After radon decays, the long lived naturally occurring radon progeny Pb being disposed and introduced to the system. Lead ions induce PW17 to form a stable G-quadruplex, thereby inhibiting the interaction between OG and PW17 and resulting in a reduction of the fluorescence intensity. The fluorescence intensity show a good linear relationship with lead ion and the radon concentration (D), thereinto, We fitted linear regression of radon concentration in the range of 0.92–4.22 (×10⁴ Bqhm⁻³) to receive a good relationship between ΔF and the concentration of radon with the detection limit of 1963 Bqhm⁻³. This method has been successfully applied for detecting standard cumulative concentration of radon and the detection limit reached the national standard of China. This sensitive method can exclude radiation damage in field testing, furthermore, it explores a new field in biological analysis using an aptamer to detected inorganic, gaseous, and radioactive materials.     

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.     

A nanogold-quenched fluorescence duplex probe for homogeneous DNA detection based on strand displacement

A nanogold-quenched fluorescence duplex probe has been developed for lighting up homogenous hybridization assays. This novel probe is constructed from two strands of different lengths, and labeled by nanogold and a fluorophore at the long-strand 5′-end and the short-strand 3′-end, respectively. The two tags are in close contact, resulting in complete quenching of the probe fluorescence. If perfectly complemented to the nanogold-labeled strand, a long target oligonucleotide would displace the short fluorophore-labeled strand, and as a result, restore the fluorescence. By using nanogold in the probe, an extremely high quenching efficiency (99.1%) and removal of free fluorophore-labeled strand is achieved. The signal-to-noise ratio and the detection limit (50 pmol L⁻¹) of homogenous assays are therefore improved significantly, in comparison with similar probes using organic acceptors. Moreover, the probe has a great inhibition effect on hybridization to a mismatched oligonucleotide. This effect provides the assay with a high specificity, and particularly the assay has great potential in applications for discriminating variations in sequences. The assay sensitivity could be markedly enhanced by using fluorescent materials in the signal strand that are brighter and not quenched by nucleobases.     

A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid

An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a “caged” inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.     

A dual detector capillary waveguide biosensor for detection and quantification of hybridized target

We describe a novel technique for improving the sensitivity of analytical instruments based on the measurement of fluorescent intensity. Independent measurement of the Rayleigh scattered intensity component by means of a second photodetector leads to normalized data, which are independent of various experimental parameters. Incorporation of this technique into a fully automated capillary waveguide biosensor improved the instrument sensitivity by a factor of three. The technique enables quantification, as well as detection, of the hybridized target molecules.     

DNA银纳米簇在功能核酸荧光生物传感器中的应用

DNA银纳米簇(DNA-AgNCs)是以DNA为模板, 通过碱基杂环上的N原子与Ag⁺结合, 用NaBH₄将Ag⁺还原得到的具有荧光性质的新兴纳米探针. 由于DNA-AgNCs具有合成方法简单、 生物相容性好和荧光发射波长可调等优点, 使其在分析检测等领域具有广泛的应用. 本文对DNA-AgNCs的合成和荧光性质两个方面进行了综述, 分类总结了以DNA-AgNCs为无标记荧光探针在功能核酸荧光生物传感器方面的应用, 对其不足与应用潜力进行展望, 以期为未来的研究与应用提供借鉴.     

Separation and determination of trace uranium using a double-receptor sandwich supramolecule method based on immobilized salophen and fluorescence labeled oligonucleotide

A double-receptor sandwich supramolecule method for the separation and determination of trace uranium was proposed in this paper. One receptor is a salophen which can react with uranyl to form a uranyl-salophen complex, and another receptor is an oligonucleotide which can bind uranyl to form oligonucleotide-uranyl-salophen supramolecule. The salophen was immobilized on the surface of silica gel particles and used as the solid phase receptor for separating uranium from solution. The oligonucleotide was labeled with a fluorescent group and used as the labeled receptor for quantitatively analyzing uranium. In the procedure of separation and determination, uranyl ion was first combined with the solid phase receptor and then conjugated with the labeled receptor to form the sandwich-type supramolecule. The labeled receptor in the sandwich supramolecule was then eluted and determined by fluorescence analysis. The experimental results demonstrate that this method has a number of advantages such as high selectivity, excellent pre-concentration capability, high sensitivity, good stability and low cost. Under optimal conditions, the linear range for the detection of uranium is 0.5–30.0 ng mL⁻¹ with a detection limit of 0.2 ng mL⁻¹. The proposed method was successfully applied for the separation and determination of uranium in real samples with the recoveries of 95.0–105.5%.     

Energy resolved X-ray fluorescence imaging based on a micropattern gas detector

A simple system for energy resolved X-ray fluorescence imaging using a room temperature, 2-D sensitive Micro-Hole and Strip Plate (MHSP) operating in pure xenon is proposed. The Micro-Hole and Strip Plate is an electron multiplier with two stages of avalanche production, one of them in the holes and another one in the anode strips. The X-ray interaction via photoelectron absorption in the xenon produces a number of electrons proportional to the incoming X-ray energy. The electron cloud is, then, amplified in the two amplification stages, resulting in a charge pulse that is also proportional to the detected X-ray energy. The 2-D capability is achieved in the Micro-Hole and Strip Plate by using two orthogonal resistive lines, one connecting the anode strips on the bottom face of the Micro-Hole and Strip Plate and the other one connecting the strips structured on the Micro-Hole and Strip Plate top surface. This low cost detector has an active area of 28 × 28 mm², an intrinsic position resolution of σ∼ 125 µm, an energy resolution of about 825 eV (Full Width at Half Maximum) at 5.9 keV and a count rate capability as high as 0.5 MHz. Fluorescence images were obtained by irradiating the sample with X-rays and using a pinhole placed between the sample and the detector window. Elemental map discrimination for different samples, image amplification and detector parameters, are presented.