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.     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.     

SERS as tool for the analysis of DNA-chips in a microfluidic platform

A sequence-specific detection method of DNA is presented combining a solid chip surface for immobilisation of capture DNAs with a microfluidic platform and a readout of the chip based on SERS. The solid chip surface is used for immobilisation of different capture DNAs, where target strands can be hybridised and unbound surfactants can be washed away. For the detection via SERS, short-labelled oligonucleotides are hybridised to the target strands. This technique is combined with a microfluidic platform that enables a fast and automated preparation process. By applying a chip format, the problems of sequence-specific DNA detection in solution phase by means of SERS can be overcome. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.     

Detection of an aberrant methylation of CDH4 gene in PCR product by ferrocenylnaphthalene diimide-based electrochemical hybridization assay

Hybridization behavior of 24-meric and 105-meric single stranded DNAs derived from CDH4 gene related to cadherin cell-adhesive protein was tested with 24-meric DNA probe in a ferrocenylnaphthalene diimide (FND)-based hybridization assay. Hybridization efficiency in this system was also clarified using chronocoulometric (CC) measurement with Hexaammineruthenium (III) probe (RuHx). This is first example to calculate hybridization efficiency of PCR product with a DNA probe immobilized on the electrode. Although hybridization efficiency was really small for the PCR product as expected (20% for 105-meric PCR product), PCR products carrying aberrant methylation were discriminated from the wild one due to the electrochemical signal of FND. It was possible since FND possessed high preference for double stranded DNA, especially on the electrode. When applied to aberrant methylation detection for the fragment of CDH4 gene, this system can discriminate over 0.5 ng μL⁻¹ sample DNA, which is superior to bisulfite sequencing or MSP and COBRA assays.     

Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization

Presently, there is a growing interest in the development of lateral flow devices for nucleic acid analysis that enable visual detection of the target sequence (analyte) while eliminating several steps required for pipetting, incubation, and washing out the excess of reactants. In this paper, we present, for the first time, lateral flow tests exploiting oligonucleotide-functionalized and antibody-functionalized carbon nanoparticles (carbon nano-strings, CBNS) as reporters that enable confirmation of the target DNA sequence by hybridization. The CBNS reporters were applied to (a) the detection of PCR products and (b) visual genotyping of single nucleotide polymorphisms in human genomic DNA. Biotinylated PCR product was hybridized with a dA-tailed probe. In one assay configuration, the hybrid is captured at the test zone of the strip by immobilized streptavidin and detected by (dT) ₃₀ -CBNS. In a second configuration, the hybrids are captured from immobilized (dA) strands and detected by antibiotin-CBNS. As low as 2.5 fmol of amplified DNA can be detected. For visual genotyping, allele-specific primers with a 5′ oligo(dA) segment are extended by DNA polymerase with a concomitant incorporation of biotin moieties. Extension products are detected either by (dT) ₃₀ -CBNS or by antibiotin-CBNS. Only three cycles of extension reaction are sufficient for detection. No purification of the PCR products or the extension product is required.     

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.     

Surface-enhanced Raman scattering detection of DNAs derived from virus genomes using Au-coated paramagnetic nanoparticles

A magnetic capture-based, surface-enhanced Raman scattering (SERS) assay for DNA detection has been developed which utilizes Au-coated paramagnetic nanoparticles (Au@PMPs) as both a SERS substrate and effective bioseparation reagent for the selective removal of target DNAs from solution. Hybridization reactions contained two target DNAs, sequence complementary reporter probes conjugated with spectrally distinct Raman dyes distinct for each target, and Au@PMPs conjugated with sequence complementary capture probes. In this case, target DNAs were derived from the RNA genomes of the Rift Valley Fever virus (RVFV) or West Nile virus (WNV). The hybridization reactions were incubated for a short period and then concentrated within the focus beam of an interrogating laser by magnetic pull-down. The attendant SERS response of each individually captured DNA provided a limit of detection sensitivity in the range 20-100 nM. X-ray diffraction and UV-vis analysis validated both the desired surface plasmon resonance properties and bimetallic composition of synthesized Au@PMPs, and UV-vis spectroscopy confirmed conjugation of the Raman dye compounds malachite green (MG) and erythrosin B (EB) with the RVFV and WNV reporter probes, respectively. Finally, hybridization reactions assembled for multiplexed detection of both targets yielded mixed MG/EB spectra and clearly differentiated peaks which facilitate the quantitative detection of each DNA target. On the basis of the simple design of a single-particle DNA detection assay, the opportunity is provided to develop magnetic capture-based SERS assays that are easily assembled and adapted for high-level multiplex detection using low-cost Raman instrumentation.     

Selective immobilization of double stranded DNA on a gold surface through threading intercalation of a naphthalene diimide having dithiolane moieties

Newly synthesized naphthalene diimide 1 having two dithiolane moieties at its substituted termini bound to double stranded DNA by threading intercalation and the resulting complex was immobilized on the gold surface through a dithiolane-gold linkage as revealed by quartz crystal microbalance (QCM) experiments. DNA with 20-meric double stranded and 24-meric single stranded regions was indirectly immobilized on the gold electrode using this characteristic of 1. Hybridization efficiency was 92%, a value higher than 50% for a thiolated oligonucleotide under identical conditions. When this electrode was subjected to hybridization with a 124-meric target DNA in the presence of ferrocenylnaphthalene diimide (FND) as an electrochemical hybridization indicator, a large current increase was observed deriving from FND bound in the double stranded region newly formed between the probe and target DNA.     

Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction

In order to develop a highly sensitive and selective piezoelectric transducer for the detection of DNA, the bio-recognizing probe is for the first time designed by introducing a hairpin structure and a recognition site for EcoRI into an oligonucleotide sequence and signal amplifiers are prepared by modifying gold nanoparticles (GNPs) with biomolecules, deepening the application and understanding of biomaterials. The piezoelectric transducer is prepared by immobilizing designed hairpin recognition probe onto the quartz-crystal-microbalance (QCM). In the absence of target DNA, the hairpin probe is removed from the QCM surface after exposure to endonuclease, inhibiting the subsequent signaling reaction. In contrast, introduction of target DNA can open the hairpin probe due to the probe/target hybridization, dissociating the cleavable double-stranded portion. In this case, even if being treated with endonuclease, the integrated hairpin probe is maintained. Subsequent introduction of GNPs modified with detection probes that can hybridize to the terminal sequence of hairpin probe results in a many-folds increase of the frequency response. Utilizing the proposed transduction scheme, the reliable target DNA detection can be accomplished. The detection limit of 2 pM and dynamic response range for target DNA from 2 to 300 pM are obtained. Furthermore, single-base mismatched DNAs can be easily identified. The developed proof-of-principle of a novel piezoelectric transduction scheme is expected to establish a potential platform for the disease-associated mutation analysis and DNA hybridization detection in biotechnology and medical diagnostics.     

DNA hybridization at microbeads with cathodic stripping voltammetric detection

In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT)(25) (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.     

玻璃微流控通道中水凝胶固定寡核苷酸探针的方法及应用

核酸杂交是分子生物学研究中最常用和最基本的分析方法之一．杂交技术有多种,主要区别在于探针的固定．目前常用的是将探针直接固定在载体表面（尼龙膜或硅烷化的玻片）或用磁珠法和水凝胶法固定,其中水凝胶法兼有三维立体和简单实用的优势,其发展颇为引人注意．微流控芯片技术具有集成化和自动化的优势．将水凝胶和微流控技术相结合,将使核酸分析中的杂交、变性以及重新杂交等操作更为简单、快速、易行．     

Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification

A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.     

A Subtractively Optimized DNA Microarray Using Non-sequenced Genomic Probes for the Detection of Food-Borne Pathogens

In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.     

Surface plasmon resonance biosensor for genetically modified organisms detection

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor.     

Electrochemiluminescent aptamer biosensor for the determination of ochratoxin A at a gold-nanoparticles-modified gold electrode using N-(aminobutyl)-N-ethylisoluminol as a luminescent label

A highly selective electrochemiluminescent biosensor for the detection of target nephrotoxic toxin, ochratoxin A (OTA), was developed using a DNA aptamer as the recognition element and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as the signal-producing compound. The electrochemiluminescent aptamer biosensor was fabricated by immobilizing aptamer complementary DNA 1 sequence onto the surface of a gold-nanoparticle (AuNP)-modified gold electrode. ABEI-labeled aptamer DNA 2 sequence hybridized to DNA 1 and was utilized as an electrochemiluminescent probe. A decreased electrochemiluminescence (ECL) signal was generated upon aptamer recognition of the target OTA, which induced the dissociation of DNA 2 (ABEI-labeled aptamer electrochemiluminescent probe) from DNA 1 and moved it far away from the electrode surface. Under the optimal conditions, the decreased ECL intensity was proportional to an OTA concentration ranging from 0.02 to 3.0 ng mL^-1, with a detection limit of 0.007 ng mL^-1. The relative standard deviation was 3.8% at 0.2 ng mL^-1 (n = 7). The proposed method has been applied to measure OTA in naturally contaminated wheat samples and validated by an official method. This work demonstrates the combination of a highly binding aptamer with a highly sensitive ECL technique to design an electrochemiluminescent biosensor, which is a very promising approach for the determination of small-molecule toxins.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

Electrochemical microfluidic biosensor for the detection of nucleic acid sequences

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.     

Electrochemical Aberrant Methylation Detection Based on Ferrocenyl Naphthalene Diimide Carrying β‐Cyclodextrin,FNC

Electrochemical detection of the specific gene carrying aberrant methylated cytosine was achieved by ferrocenyl naphthalene diimide carrying β‐cyclodextrin (β‐CD), FNC, coupled with the probe‐DNA‐immobilized electrode. The five CpG sites in a 24‐base sequence were selected as the target DNA on the CDH4 gene, which is associated with colorectal cancer. When methylated and unmethylated samples hybridized with the DNA probe (HS‐M24) immobilized on the electrode, an increased current signal was observed in the electrolyte containing FNC and correlated with the amount of target DNA. Furthermore, an increase in current (115 %) was observed when the PCR product of 105 bp was hybridized on the HS‐M24‐immobilized electrode, whereas a background level of current increase was observed in the case of unmethylated product. Such large discrimination ability might be due to the inter‐ and/or intra‐complex formation of ferrocene with β‐CD of FNC on the surface of the electrode.