Spectrofluorometry of Ions and Small Molecules Based on Conformational Changes of Specific Oligonucleotides with o-Phthalaldehyde-β-mercaptoethanol

Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol(OPAME) has been extensively applied to fluorescent detection of amino compounds based on the reaction of primary amino-group, herein we proposed a general spectrofluorometry for ions and small molecules due to conformational changes upon ligand binding taking K+ and ATP as examples. In a borate controlled buffer medium, telomere DNA could react with OPAME, giving a thio-subtituted isoindole compound with strong fluorescence emission at 455 nm when excited at 340 nm. It was found that however, the fluorescence emission was greatly reduced in the presence of K+ since the formation of the quadruplex structure inhibits the reaction activity of amino-groups of telomere DNA. In order to testify the general application of OPAME reagent based on the conformational change of oligonucleotides, we further proposed a sensitive method of ATP based on its highly selective interaction with ATP-aptamer. The above mentioned applications show that the spectrofluorometry with the aid of OPAME reagent is simple, label free that is expected to be potentially general for DNA conformational change-based target detection.     

水溶性碳纳米粒子的制备及其在DNA和单核苷酸多态性分析中的应用

利用电化学氧化的方法制备了水溶性好、粒径为7～12nm的碳纳米粒子,该碳纳米粒子通过π-π相互作用吸附荧光标记的单链DNA探针,并能有效地猝灭其荧光．当单链DNA探针与匹配的DNA目标分子杂交形成双链DNA时,猝灭的荧光被恢复,由此可以检测1-200nmol／L的DNA目标分子。此外,在碳纳米粒子存在时,由荧光标记的DNA探针和DNA目标分子形成的双链DNA的熔解温度可以简便地被测定,当双链DNA有错配碱基时,其熔解温度降低,由此可方便、快速地分析单核苷酸多态性．     

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

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

基于银纳米粒子构建荧光传感平台用于核酸检测

报道了基于银纳米粒子构建的荧光传感平台,并用于核酸检测.此荧光传感平台对核酸检测基于以下策略:首先,荧光团标记的单链DNA探针被吸附到银纳米粒子的表面,荧光团与银纳米粒子近距离接触,发生荧光猝灭;加入与探针DNA序列互补的目标DNA,两者杂交形成双链DNA,并从银纳米粒子的表面脱离,荧光得到恢复.这种银纳米粒子构建的荧...     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

Reaction kinetics study of short DNA strands using a maximum entropy method and nonlinear curve fitting

It is important to understand the formation of double-strand DNA (dsDNA) in a salt solution because it is one of the key reactions in life. A short cDNA strand pair was designed, and each single-strand DNA (ssDNA) was attached to a fluorescent dye that was either a donor or an acceptor of fluorescence resonance energy transfer. The fluorescence intensity was expected to change as time passed as the complementary pairs of ssDNAs formed dsDNAs. The concentration of dsDNA was theoretically calculated, and the measured data were consistent with theoretical results. The analysis of the nonlinear fitting method and the maximum entropy method detected that the reaction curve contains two major types of kinetics that likely represent the formation of dsDNA and mismatching.     

Application of 3,4,9,10-perylenetetracarboxylic diimide microfibers as a fluorescent sensing platform for biomolecular detection

In this paper, we report on the use of 3,4,9,10-perylenetetracarboxylic diimide microfibers (PDIMs) as an effective fluorescent sensing platform for DNA detection for the first time. This sensing system exhibits a detection limit as low as 15 nmol L⁻¹ and has a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of fluorescently labeled single-stranded DNA (ssDNA) probe by PDIM due to the strong π–π stacking between unpaired DNA bases and PDIM. As a result, the fluorophore is brought into close proximity of PDIM, leading to substantial fluorescence quenching. In the presence of the target, the specific hybridization of the probe with its complementary DNA sequence generates a double stranded DNA (dsDNA) which detaches from PDIM, leading to fluorescence recovery. Its generality of this sensing platform for protein detection is also demonstrated.     

Non-covalent anionic porphyrin functionalized multi-walled carbon nanotubes as an optical probe for specific DNA detection

With water-soluble anionic tetra (p-carboxyphenyl) porphyrin (TCPP) to solubilize multi-walled carbon nanotubes (MCNTs), we obtained a suspension that could be stable more than 1 week. With this TCPP/MCNTs suspension, we propose a spectrofluorometric method of DNA hybridization in this contribution. Our basic finding for this work is that the fluorescence from a dye-tagged single stranded DNA (ssDNA), which was directly added to the TCPP/MCNTs suspension, gets quenched, and the fluorescence could be remained if the dye-tagged single stranded DNA is first to be hybridized with its complementary target DNA to form a double stranded DNA (dsDNA) hybrid and added into the TCPP/MCNTs suspension. Mechanism investigations showed that the reason for the former is due to the adsorption of ssDNA on the surfaces of MCNTs, and that for the latter is due to the strong electrostatic repulsion force between the negative charge TCPP/MCNTs complexes and dsDNA. Thus, target DNA in a DNA sample and single-base mismatch in DNA sequences could be easily detected.     

A facile graphene oxide-based DNA polymerase assay

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.     

Supramolecular Reassembly of Self‐Exfoliated Ionic Covalent Organic Nanosheets for Label‐Free Detection of Double‐Stranded DNA

Ionic covalent organic nanosheets (iCONs), a member of the two‐dimensional (2D) nanomaterials family, offer a unique functional platform for a wide range of applications. Herein, we explore the potential of an ethidium bromide (EB)‐based covalent organic framework ( EB‐TFP ) that self‐exfoliates in water resulting in 2D ionic covalent organic nanosheets ( EB‐TFP‐iCONs ) for the selective detection of double‐stranded DNA (dsDNA). In an aqueous medium, the self‐exfoliated EB‐TFP‐iCONs reassemble in the presence of dsDNA resulting in hybrid EB‐TFP‐iCONs‐DNA crystalline nanosheets with enhanced fluorescence at 600 nm. Detailed steady‐state and time‐resolved emission studies revealed that the reassembly phenomenon was highly selective for dsDNA when compared to single‐stranded DNA (ssDNA), which allowed us to use the EB‐TFP‐iCONs as a 2D fluorescent platform for the label‐free detection of complementary DNA strands.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法

建立了一种基于阳离子型共轭聚合物和核酸适体的腺苷检测新方法. 荧光素修饰的短链DNA与腺苷的核酸适体部分互补, 形成双链DNA; 阳离子型共轭聚合物通过静电作用与双链DNA结合, 发生高效率的荧光共振能量转移(FRET). 加入腺苷后, 腺苷与核酸适体发生特异性结合, 导致双链DNA分解成单链, 使静电吸引力下降, 能量转移效率降低. 通过阳离子型共轭聚合物对单双链DNA的高效识别, 可快速简易地检测出腺苷.     

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.     

Formation of a DNA layer on Langmuir-Blodgett films and its enzymatic digestion

Here, we report a system we have developed where long double-stranded DNAs (dsDNAs) are immobilized on a monolayer of Zn-arachidate. We have applied the Langmuir-Blodgett technique to form the monolayer of Zn-arachidate where Zn(II) is bound to arachidic acid through charge neutralization. Because tetrahedral Zn(II) participates in DNA recognition through coordination, we have been able to layer DNA over the Zn-arachidate monolayer. The DNA layer shows a typical compression and expansion cycle in a concentration-dependent fashion. Interestingly, the DNA monolayer is available for enzymatic degradation by DNaseI. The detection of DNA and its accessibility towards biological reaction is demonstrated by imaging through fluorescence microscopy. The conformation of the DNA, immobilized on the monolayer, was studied with the help of atomic force microscopy (AFM). We observed that the dsDNAs were aligned in a stretched manner on the surface. To investigate further, we also demonstrate here that the small single-stranded DNA (ssDNA) immobilized on the air-water interface can act as a target molecule for the complementary ssDNA present in the subphase. The study of DNA hybridization done with the help of fluorescence spectroscopy clearly supports the AFM characterization.     

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.     

Acridine-viologen dyads: selective recognition of single-strand DNA through fluorescence enhancement

Tolylacridine-viologen dyads show distinct fluorescence emission changes in the presence of double-strand DNA (dsDNA) and single-strand DNA (ssDNA) depending on the position of the linkage. The para isomer shows fluorescence quenching in the presence of both dsDNA and ssDNA, while the ortho isomer interacts selectively with ssDNA with enhancement in fluorescence intensity.     

Selective enzymatic labeling to detect packing-induced denaturation of double-stranded DNA at interfaces

The adsorption of DNA on surfaces is a widespread procedure and is a common way for fabrication of biosensors, DNA chips, and nanoelectronic devices. Although the biologically relevant and prevailing in vivo structure of DNA is its double-stranded (dsDNA) conformation, the characterization of DNA on surfaces has mainly focused on single-stranded DNA (ssDNA). Studying the structure of dsDNA on surfaces is of invaluable importance to microarray performance since their effectiveness relies on the ability of two DNA molecules to hybridize and remain stable. In addition, many of the enzymatic transactions performed on DNA require dsDNA, rather than ssDNA, as a substrate. However, it is not established that adsorbed dsDNA remains in its structure and does not denature. Here, two methodologies have been developed for distinguishing between surface-adsorbed single- and double-stranded DNA. We demonstrate that, upon formation of a dense monolayer, the nonthiolated strand comprising the dsDNA is released and the monolayer consists of mostly ssDNA. The fraction of dsDNA within the ssDNA monolayer depends on the length of the oligomers. A likely mechanism leading to this rearrangement is discussed.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

Programmable and Reversible Regulation of Catalytic Hemin@MOFs Activities with DNA Structures

Metal-organic frameworks(MOFs)-based nanozyme plays an important role in biosensing,therapy and catalysis.In this study,the effects of single-stranded DNA(ssDNA)with programmable sequences and its complementary DNA(Tdna)on the intrinsic peroxidase-like activity of hemin loaded MOFs(UiO-66-NH2),denoted as he-min@UiO-66-NH2,were investigated.The hemin@UiO-66-NH2 exhibited improved catalytic activity compared with free hemin.However,the catalytic activity is inhibited in the presence of ssDNA,as ssDNA can be adsorbed by MOFs and therefore protected the active sites from contact with substrates.Upon the addition of the TDNA,double-stranded DNA(dsDNA)was formed and detached from the MOFs,resulting in the recovery of catalytic activity.Sequentially adding ssDNA or its complementary DNA strands can achieve the reversible regulation of the catalytic activity of MOFs nanozymes.Moreover,the DNA hybridization-based regulation was further applied to a cascaded catalytic system composed of the nanozyme,hemin@UiO-66-NH2,and glucose oxidase.These nanozyme based programmable and reversibly regulated catalytic systems may have potential applications in future smart biosensing and catalysis systems.