Unlabeled hairpin-DNA probe for the detection of single-nucleotide mismatches by electrochemical impedance spectroscopy

An unlabeled hairpin-DNA probe was used for the detection of eight single-nucleotide mismatches by electrochemical impedance spectroscopy (EIS). Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened and forms a duplex DNA. Accordingly, the film thickness is increased, which causes differences in the electrical properties of the film before and after hybridization. Randles equivalent circuits were employed to evaluate the EIS result. The differences in the charge-transfer resistance DeltaR(CT) between hairpin DNA (before hybridization) and duplex DNA (after hybridization) shows the consequence of a large structural rearrangement from hairpin to duplex. If a single-nucleotide mismatch is present in the center of the duplex, the difference in charge-transfer resistance DeltaR(CT) between B-DNA in the absence and presence of Zn(2+) allows the unequivocal detection of all eight single-nucleotide mismatches. The detection limit was measured, and DeltaR(CT) allows the discrimination of a single-nucleotide mismatch with the concentration of the target strand as low as 10 pM.     

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.     

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.     

Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay

MicroRNAs (miRNAs) play important regulatory roles in a wide range of biological processes, and their aberrant expression is associated with cancer development and a variety of diseases. Here, we develop a simple, sensitive, and specific miRNA assay on the basis of circular exponential amplification in combination with the hairpin probes. The binding of target miRNA with a linear DNA template initiates the first strand displacement amplification (SDA) and generates the universal triggers which are complementary to the 3' protruding end of a hairpin probe. These universal triggers function not only as the primers to unfold the hairpin probes through an extension reaction, generating distinct fluorescence signals, but also as the amplification templates to initiate the second SDA reaction. Moreover, the second SDA reaction can release new triggers to initiate the above two consecutive SDA reactions, thus constituting a circular exponential amplification which enables the conversion of a small amount of miRNAs to a large number of universal triggers to unfold abundant hairpin probes. This hairpin probe-based circular exponential amplification assay exhibits high sensitivity with a detection limit of 3.80 × 10(-13) M and a detection range of 4 orders of magnitude. It can even discriminate single-nucleotide difference between miRNA family members and perform well in real sample analysis. Notably, in this assay, the long-stem hairpin probes are unfolded through an extension reaction rather than through a conventional hybridization reaction controlled by the thermodynamic equilibrium in the case of molecular beacons, making the design of hairpin probes very simple. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.     

Conformational switching immobilized hairpin DNA probes following subsequent expanding of gold nanoparticles enables visual detecting sequence-specific DNA

A simple, rapid, and sensitive method for visual detection of sequence-specific DNA was developed using hairpin DNA as the recognition element and hydroxylamine-enlarged gold nanoparticles (Au-NPs) as the signal producing component. In the assay, we employed a hairpin DNA probe dually labeled with amine and biotin at the 5'- and 3'-end, respectively. The probe was coupled with reactive N-oxysuccinnimide in a DNA-bind 96-well plate. Without the target DNA, the immobilized hairpin probe was in a "closed" state, which kept the streptavidin-gold off the biotin. The hybridization between the loop sequence and the target broke the short stem duplex upon approaching the target DNA. Consequently, biotin was forced away from the 96-well plate surface and available for conjugation with the streptavidin-gold. The hybridization could be detected visually after the HAuCl(4)-NH(2)OH redox reaction catalyzed by the Au-NPs. Under the optimized conditions, the visual DNA sensor could detect as low as 100 amol of DNA targets with excellent differentiation ability and even a single-base mismatch.     

"Off-On" Electrochemical Hairpin-DNA-Based Genosensor for Cancer Diagnostics

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.     

DNA covalent immobilization onto screen-printed electrode networks for direct label-free hybridization detection of p53 sequences

A new electrochemical biochip for the detection of DNA sequences was developed. The entire biochip-i.e., working, reference, and counter electrodes-was constructed based on the screen-printing technique and exhibits eight working electrodes that could be individually addressed and grafted through a simple electrochemical procedure. Screen-printed electrode networks were functionalized electrochemically with 1-ethyl-3-(3dimethylaminopropyl)carbodidiimide according to a simple procedure. Single-stranded DNA with a C6-NH(2) linker at the 5'-end was then covalently bound to the surface to act as probe for the direct, nonlabeled, detection of complementary strands in a conductive liquid medium. In the present system, the study was focused on a particular codon (273) localized in the exon 8 of the p53 gene (20 mer, TTGAGGTGCATGTTTGTGCC). The integrity of the immobilized probes and its ability to capture target sequences was monitored through chemiluminescent detection following the hybridization of a peroxidase-labeled target. The grafting of the probe at the electrode surface was shown to generate significant shifts of the Nyquist curves measured in the 10-kHz to 80-Hz range. These variations of the faradaic impedance were found to be related to changes of the double layer capacitance of the electrochemical system's equivalent circuit. Similarly, hybridization of complementary strands was monitored through the measurements of these shifts, which enabled the detection of target sequences from 1 to 200 nM. Discrimination between complementary, noncomplementary, and single-nucleotide mismatch targets was easily accomplished.     

Direct detection of DNA conformation in hybridization processes

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

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.     

Microenvironmental properties and chiral discrimination abilities of bile salt micelles by fluorescence probe technique

The microenvironmental properties as well as the chiral discrimination abilities of four kinds of bile salt micelles were investigated by the fluorescence probe technique. A new fluorescence probe, 1,1'-bi-2-naphthol, was exploited to study the aggregation of bile salts, size, micropolarity, and microrigidity of the micelles with or without the presence of inorganic salts. Based on these results, the chiral discrimination abilities of bile salt micelles were further investigated by using (R)- and (S)-1,1'-bi-2-naphthol as chiral fluorescence probes. Different chiral discrimination ability was revealed by fluorescence spectra, fluorescence increase rate, and fluorescence quenching constants. The chiral discrimination mechanism of bile salt micelles was discussed.     

Evaluating the binding affinities of NF-kappaB protein to the single-nucleotide mismatch DNA binding sites by using double-stranded DNA microarray

Protein-DNA sequence-specific interaction plays an essential role in many biological processes. Here we immobilized a series of double-stranded DNA probes on an agarose coated slide to investigate the binding affinity of NF-kappaB p50 homodimer to the single-nucleotide mismatches (G<-->A or T<-->C) of the 10 base pair (bp) protein binding sites. The results demonstrated that the nucleotides at different positions contribute differently to the p50p50/DNA binding interaction. Within the 10 bp binding sites, the 5tG or 6cA mismatch has less effect on the protein-DNA binding affinity. Even the 5tG mismatch may have the ability to enhance the protein-DNA interaction (5t/w = 1.07). On the other hand, the 7cA or 10tG mismatch blocked the protein-DNA interaction more significantly than other six single-nucleotide mismatches. (7c/W = 0.37, 10t/W = 0.35). It also indicated that the duplex DNA probes immobilized on the agarose-coated surface were apt to be recognized by DNA-binding proteins, and this method would provide a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.     

Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of Lamivudine resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.     

Microfabricated linear hydrogel microarray for single-nucleotide polymorphism detection

A platform is developed for rapid, multiplexed detection of single-nucleotide polymorphisms using gels copolymerized with oligonucleotide capture probes in a linear microchannel array. DNA samples are analyzed by electrophoresis through the linear array of gels, each containing 20-40 μM of a unique oligonucleotide capture probe. Electrophoresis of target DNA through the capture sites and the high concentration of capture probes within the gels enables significantly shorter incubation times than standard surface DNA microarrays. These factors also result in a significant concentration of target within the gels, enabling precise analysis of as little as 0.6 femtomoles of DNA target. Differential melting of perfectly matched and mismatched targets from capture probes as a function of electric field and temperature enables rapid, unambiguous identification of single-nucleotide polymorphisms.     

Structure and DNA hybridization properties of mixed nucleic acid/maleimide-ethylene glycol monolayers

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HS-ssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Orientation of the ssDNA probes was determined by near-edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s --> pi* transition) indicate that the immobilized ssDNA molecules reorient toward a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high-density" probe surface than on the "high-efficiency" probe surface. The amounts of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to nonspecific serum protein adsorption onto the sensing surface.     

Fabrication and application of single nucleotide polymorphisms library on magnetic nanoparticles using adaptor PCR

We have developed a novel approach to fabricate single nucleotide polymorphisms (SNPs) library on magnetic nanoparticles (MNPs) based on adaptor PCR. Each SNP locus in the library was interrogated by hybridization with a pair of allele specific dual-color fluorescence (Cy3, Cy5) probes to determine SNP. Two SNPs loci (M235T and A-6G) associated with essential hypertension in the angiotensinogen (AGT) gene were detected by this method and their fluorescent signals were quantified. The fluorescent ratios (match probe: mismatch probe signal) of homozygous genotypes were over 3.0, whereas heterozygous genotypes had ratios near to 1.0. Without any complex multiplex PCR procedure, it is a simple, efficient and reliable method for the multiplex SNPs detection using limited amount of DNA samples from individuals.     

Enzymatic on-chip enhancement for high resolution genotyping DNA microarrays

Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant Klebsiella pneumoniae carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination.     

基于DNA荧光探针的T4多聚核苷酸激酶 免标记荧光检测

开发一种新型的基于DNA荧光探针的T4多聚核苷酸激酶(T4 PNK)活性检测方法。先设计了可以形成发卡结构的DNA探针(PNK-Tb),再通过引入T4 PNK、ATP和λ核酸外切水解酶(λ exo),打开发卡结构,释放发卡结构3’末端富含G的碱基序列,随后与Tb~(3+)结合形成G-四链体产生显著的荧光信号。通过反应前后荧光信号的变化实现T4 PNK的高灵敏检测。实验结果表明:成功制备了新的免标记DNA荧光探针,创新性地将Tb~(3+)应用到T4 PNK活性的检测中;本荧光法定量检测的线性范围为0~100 U/mL,检测下限为2 U/mL;该策略具有良好的特异性并且可用于评估ADP对T4 PNK活性的抑制作用。基于免淬灭标记DNA荧光探针构建的T4 PNK活性检测新策略反应快速 (不超过60 min)、成本低廉、灵敏度高,在药物开发以及生物化学研究中具有广阔的应用前景。     

Dynamic DNA hybridization on a chip using paramagnetic beads

Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.     

Monitoring molecular beacon/DNA interactions using atomic force microscopy

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.     

Study of single-stranded DNA binding protein-nucleic acids interactions using unmodified gold nanoparticles and its application for detection of single nucleotide polymorphisms

We have developed a label-free homogeneous phase bioassay to characterize the DNA binding properties of single-stranded DNA binding (SSB) protein, a key protein involved in various DNA processes such as DNA replication and repair. This assay uses gold nanoparticles (AuNPs) as sensing probe and is based on the phenomenon that preformed SSB-single-stranded DNA (ssDNA) complexes can protect AuNPs against salt-induced aggregation better than SSB or ssDNA alone. With the controlled particle aggregation/dispersion as measure, this assay can be used to detect the formation of SSB complexes with ssDNA of different length and nucleotide composition and to assess their binding properties without tedious and complicated assay procedures. On the basis of the inverse relationship between DNA hybridization efficiency and the tendency of SSB to form protection complex with unhybridized ssDNA to AuNPs, this assay is further developed to detect DNA hybridization with single nucleotide polymorphism selectivity. Owing to the high affinity between SSB and dissociated ssDNA, single-base mismatch discrimination in a long sequence of 30-mer DNA was achieved for both the end- and center-base mismatch. Unlike the conventional techniques for DNA and protein analysis, current AuNPs-based sensing strategy is simple in design, fast in detection, and economical for operation without the need of sophisticated equipment.