Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5'-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3'(HP) containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.     

基于链置换循环无标记检测端粒酶RNA的荧光法

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂, SYBR Green Ⅰ(SGⅠ)为荧光信号探针, 发夹核酸探针为分子识别探针, 基于目标物启动的发夹核酸探针链置换循环反应, 建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA(hTR)的荧光新方法. 发夹核酸探针hpDNA1和hpDNA2吸附在GO表面, 嵌插在发夹DNA探针茎部的SGⅠ的荧光信号被GO猝灭. 当人工合成的目标物(T1)存在时, T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应, 由此累积产生大量的hpDNA1/hpDNA2杂交双链. 刚性的双链DNA脱离GO表面, 导致所嵌插的SGⅠ产生较强的荧光信号. 基于荧光信号的变化, 可定量检测0.2~50 nmol/L的T1, 检出限为90 pmol/L. 该方法为端粒酶RNA检测提供了一种高灵敏、 高特异性且无需标记的荧光新途径.     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

Enzyme-free and multiplexed microRNA detection using microRNA-initiated DNA molecular motor

In this work, we have developed a sensitive, simple, and enzyme-free assay for detection of microRNAs (miRNAs) by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains. In the presence of miRNA target, it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand (ss) DNA, the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand (ds) DNA to release the miRNA. One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence. The formation of dsDNA can produced specific fluorescence signal for miRNA detection. The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor, which results in great fluorescence amplification. With the efficient signal amplification, as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained. Moreover, by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores, multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum (SFS) technique.     

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.     

Dynamic-light-scattering-based sequence-specific recognition of double-stranded DNA with oligonucleotide-functionalized gold nanoparticles

An ultrasensitive and simple dynamic-light-scattering (DLS) assay for the sequence-specific recognition of double-stranded DNA (dsDNA) was developed based on detection of the average diameter change of Au nanoparticle (AuNP) probes modified with oligonucleotides 5'-TTTCTCTTCCTT- CTCTTC-(T)(12)-SH-3' (Oligo 1) and 5'-TTCTTTCTTTTCTTTTTC-(T)(12)- SH-3' (Oligo 2). The target dsDNA was composed of two complementary oligonucleotides: 5'-AAAGAGAAGGAAGAGAAGAAGAAAGAAAAGAAAAAG-3' (Oligo 3) and 3'-TTTCTCTTCCTTCTCTTCTTCTTTCTTTTCTTTTTC-5' (Oligo 4). Hybridization of the two AuNPs-Oligo probes with the target dsDNA induced aggregation of the target dsDNA by forming triplex DNA, which accordingly increased the average diameter. This diameter change could then be detected by DLS. The average diameter was proportional to the target dsDNA concentration over the range from 593 fM to 40 pM, with a detection limit of 593 fM. Moreover, the assay had good sequence specificity for the target dsDNA.     

Site-specific detection of DNA methylation utilizing mCpG-SEER

Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.     

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.     

A Competitor-switched Electrochemical Sensor for Detection of DNA

A competitor‐switched electrochemical sensor based on a generic displacement strategy was designed for DNA detection. In this strategy, an unmodified single‐stranded DNA (cDNA) completely complementary to the target DNA served as the molecular recognition element, while a hairpin DNA (hDNA) labeled with a ferrocene (Fc) and a thiol group at its terminals served as both the competitor element and the probe. This electrochemical sensor was fabricated by self‐assembling a dsDNA onto a gold electrode surface. The dsDNA was pre‐formed through the hybridization of Fc‐labeled hDNA and cDNA with their part complementary sequences. Initially, the labeled ferrocene in the dsDNA was far from surface of the electrode, the electrochemical sensor exhibited a "switch‐off" mode due to unfavorable electron transfer of Fc label. However, in the presence of target DNA, cDNA was released from hDNA by target DNA, the hairpin‐open hDNA restored its original hairpin structure and the ferrocene approached onto the electrode surface, thus the electrochemical sensor exhibited a "switch‐on" mode accompanying with a change in the current response. The experimental results showed that as low as 4.4×10⁻¹⁰ mol/L target DNA could be distinguishingly detected, and this method had obvious advantages such as facile operation, low cost and reagentless procedure.     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

Colloidal gold-polystyrene bead hybrid for chemiluminescent detection of sequence-specific DNA

A sensitive chemiluminescent (CL) detection of sequence-specific DNA has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold labels. In this protocol, the target oligonucleotides are hybridized with magnetic bead-linked capture probes, followed by the hybridization of the biotin-terminated amplifying DNA probes and the binding of streptavidin-coated gold nanoparticles; the nanometer-sized gold tags are then dissolved and quantified by a simple and sensitive luminol CL reaction. The proposed CL protocol is evaluated for a 30-base model DNA sequence, and the amount as low as 0.01 pmol of DNA is determined, which exhibits a 150 x enhancement in sensitivity over previous gold dissolution-based electrochemical formats and an enhancement of 20 x over the ICPMS detection. Further signal amplification is achieved by the assembly of biotinylated colloidal gold onto the surface of streptavidin-coated polystyrene beads. Such amplified CL transduction allows detection of DNA targets down to the 100 amol level, and offers great promise for ultrasensitive detection of other biorecognition events.     

Ultrasensitive and Signal‐on Electrochemiluminescence Aptasensor Using the Multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin Complexes

An ultrasensitive and signal‐on electrochemiluminescence (ECL) aptasensor to detect target protein (thrombin or lysozyme) was developed using the host‐guest recognition between a metallocyclodextrin complex and single‐stranded DNA (ss‐DNA). The aptasensor uses both the photoactive properties of the metallocyclodextrins named multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin complexes and their specific recognition with ss‐DNA, which amplified the ECL signal without luminophore labeling. After investigating the ECL performance of different multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin (multi‐Ru‐β‐CD) complexes, tris‐tris(bipyridine)‐ruthenium(II)‐β‐cyclodextrin (tris(bpyRu)‐β‐CD) was selected as a suitable host molecule to construct an atasensor. First, double‐stranded DNA (ds‐DNA) formed by hybridization of the aptamer and its target DNA was attached to a glassy carbon electrode via coupling interaction, which showed low ECL intensity with 2‐(dibutylamino) ethanol (DBAE) as coreactant, because of the weak recognition between ds‐DNA and tris(bpyRu)‐β‐CD. Upon addition of the corresponding protein, the ECL intensity increased when target ss‐DNA was released because of the higher stability of the aptamer‐protein complex than the aptamer‐DNA one. A linear relationship was observed in the range of 0.01 pmol/L to 100 pmol/L between ECL intensity and the logarithm of thrombin concentrations with a limited detection of 8.5 fmol/L (S/N=3). Meanwhile, the measured concentration of lysozyme was from 0.05 pmol/L to 500 pmol/L and the detection limit was 33 fmol/L (S/N=3). The investigations of proteins in human serum samples were also performed to demonstrate the validity of detection in real clinical samples. The simplicity, high sensitivity and specificity of this aptasensor show great promise for practical applications in protein monitoring and disease diagnosis.     

Label-free and sequence-specific DNA detection down to a picomolar level with carbon nanotubes as support for probe DNA

This study describes a simple and label-free electrochemical impedance spectroscopic (EIS) method for sequence-specific detection of DNA by using single-walled carbon nanotubes (SWNTs) as the support for probe DNA. SWNTs are confined onto gold electrodes with mixed self-assembly monolayers of thioethanol and cysteamine. Single-stranded DNA (ssDNA) probe is anchored onto the SWNT support through covalent binding between carboxyl groups at the nanotubes and amino groups at 5′ ends of ssDNA. Hybridization of target DNA with the anchored probe DNA greatly increases the interfacial electron-transfer resistance (R_et) at the double-stranded DNA (dsDNA)-modified electrodes for the redox couple of Fe(CN)₆^3−/4−, which could be used for label-free and sequence-specific DNA detection. EIS results demonstrate that the utilization of SWNTs as the support for probe DNA substantially increases the surface loading of probe DNA onto electrode surface and thus remarkably lowers the detection limit for target DNA. Under the conditions employed here, R_et is linear with the concentration of target DNA within a concentration range from 1 to 10 pM with a detection limit down to 0.8 pM (S/N = 3). This study may offer a novel and label-free electrochemical approach to sensitive sequence-specific DNA detection.     

Construction and Electrochemical Property Studies of DNA Duplexes Tethered to Gold Electrode via Au−C Bond

Effective linkage of DNA onto metal surfaces plays a crucial role in the applications of DNA as electrochemical recognition, signal output and amplification devices for gene and protein detections, specific analyte recognitions, catalysis, and so on. Here we report a promising and operationally simple approach for the construction of double‐stranded DNA‐linked Au interface via Au?C bond (RdsDNA‐C?Au), upon efficient in situ cleavage of trimethylsilyl end group of 4‐[(trimethylsilyl) ethynyl] benzoic acid and subsequent dehydration condensation between NH₂‐modified DNA and benzoic acid. Due to the introduction of large conjugated π group (4‐carboxyphenylethynyl) as the “bridge bond”, the conductivity of the RdsDNA‐C?Au interface is greatly improved. As a result, under commonly used DNA packing density (>0.5 pmol cm^?2), the surface‐confined electron transfer at the interface is simply mediated by the stacked‐bases of dsDNA, independent of the orientation of dsDNA (tethered to the electrode at 5′‐ or 3′‐end). Also, compared to the traditional RdsDNA‐S?Au interface via alkanethiol linker, the RdsDNA‐C?Au interface displays more sensitive electrochemical response and excellent stability. Due to the better stability, conductivity and simple electron transfer mechanism, the RdsDNA‐C?Au interface is anticipated to be widely used in electrochemistry‐involved molecular recognitions, gene and protein detections with higher sensitivity and accuracy.     

Ultrasensitive SERS Detection of Lysozyme by a Target‐Triggering Multiple Cycle Amplification Strategy Based on a Gold Substrate

An ultrasensitive surface enhanced Raman scattering (SERS) method has been designed to selectively and sensitively detect lysozyme. The gold chip as the detection substrate, the aptamer‐based target‐triggering cascade multiple cycle amplification, and gold nanoparticles (AuNPs) bio‐barcode Raman probe enhancement on the gold substrate are employed to enhance the SERS signals. The cascade amplification process consists of the nicking enzyme signaling amplification (NESA), the strand displacement amplification (SDA), and the circular‐hairpin‐assisted exponential amplification reaction (HA‐EXPAR). With the involvement of an aptamer‐based probe, two amplification reaction templates, and a Raman probe, the whole circle amplification process is triggered by the target recognition of lysozyme. The products of the upstream cycle (NESA) could act as the “DNA trigger” of the downstream cycle (SDA and circular HA‐EXPAR) to generate further signal amplification, resulting in the immobility of abundant AuNPs Raman probes on the gold substrate. “Hot spots” are produced between the Raman probe and the gold film, leading to significant SERS enhancement. This detection method exhibits excellent specificity and sensitivity towards lysozyme with a detection limit of 1.0×10^?15 M . Moreover, the practical determination of lysozyme in human serum demonstrates the feasibility of this SERS approach in the analysis of a variety of biological specimens.     

Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.     

Single-stranded DNA (ssDNA) production in DNA aptamer generation

The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.     

Label-free and non-enzymatic detection of DNA based on hybridization chain reaction amplification and dsDNA-templated copper nanoparticles

A label-free and non-enzymatic amplification fluorescent method for detection of DNA has been developed by using hybridization chain reaction (HCR) and dsDNA-templated copper nanoparticles (CuNPs). First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of dsDNA polymers while two hairpin probes coexisted. Subsequently, the dsDNA polymers were employed as template for synthesis of CuNPs with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. The concept and methodology developed in this work show great promise in the quantitative detection of DNA in biological and medical applications.     

Electrochemical Luminescent DNA Sensor Based on Polymerase-assisted Signal Amplification

A novel polymerase-based electrochemical luminescence (ECL) DNA sensor was constructed for messenger RNA (mRNA) detection by cyclic chain displacement polymerization, assisted by target mRNA cycle and quantum dots signal amplification. Firstly, the mercapto-modified capture-type probe DNA (CP) was immobilized on the surface of a magneto-controlled glassy carbon electrode via Au-S bond. After the addition of target mRNA, CP was opened and hybridized with mRNA to form double-stranded DNA (dsDNA). Then polymerase, primer chain (DNA1) and bases were added, which made the primer chain extend to replace the target mRNA. After one amplification cycle, the mRNA chain could open another hairpin in order to carry out next cycle of amplification. Finally, the ECL detection was carried out by adding DNA2 labeled thioglycolic acid-CdTe quantum dots. The amplification of the target mRNA by the addition of polymerase and the signal combined with the quantum dots label greatly improved the sensitivity of the sensor. The results showed that corresponding ECL signal had a good linear relationship with logarithm of target mRNA concentration in the range of 1 × 10^?15 to 1 × 10^?11 M, with a detection limit of 3.4 × 10^?16 M (S/N = 3). Under the optimal conditions, the recoveries of mRNA spiked in human serum sample were from 97.2 % to 102.3 %. This sensor exhibited good selectivity, stability and reproducibility.