Direct energy transfer from conjugated polymer to DNA intercalated dye: label-free fluorescent DNA detection

The development of methods for DNA detection is of importance in disease diagnosis, gene-targeted drug discovery and molecular biology field. In this paper, we synthesize a new cationic water-soluble CP containing fluorene moiety and flexible ethylenic moiety in the backbone (PFV) for label-free DNA detection. The conformational freedom of PFV provides stronger interactions with double-stranded DNA (dsDNA) and optimizes the orientation of transition moments between PFV and ethidium bromide (EB) intercalated in dsDNA. The efficient FRET from PFV (donor) to EB (acceptor) intercalated in dsDNA is observed and the emission of EB is amplified by the good light-harvesting ability of conjugated polymers. The interactions between PFV and DNA can also be probed by measuring the FRET ratio between PFV and EB intercalated in DNA. In comparison to other DNA detection assays based on FRET and conjugated polymers, synthesis of dye-labeled DNA probe is avoided in our method, which significantly reduces the cost and the synthetic complexity. The PFV/dsDNA/EB system provides promising applications on DNA detection with a simply, fast and label-free manner.     

Cationic conjugated polyelectrolyte/molecular beacon complex for sensitive, sequence-specific, real-time DNA detection

A new fluorescence method has been developed for DNA detection at room temperature in a sensitive, selective, economical, and real-time manner that interfaces the superiority of a molecular beacon in mismatch discrimination with the light-harvesting property of water-soluble conjugated polyelectrolytes. The probe solution contains a cationic conjugated polyelectrolyte (PFP-NMe3+), a molecular beacon with a five base pairs double-stranded stem labeled at the 5'-terminus with fluorescein (DNA P-Fl), and ethidium bromide (EB, a specific intercalator of dsDNA). The electrostatic interactions between DNA P-Fl and PFP-NMe3+ keep them in close proximity, facilitating the fluorescence resonance energy transfer (FRET) from PFP-NMe3+ to fluorescein. Upon adding a complementary strand to the probe solution, the conformation of DNA P-Fl transits into dsDNA followed by the intercalation of EB into the grooves. Two-step FRET, from PFP-NMe3+ to DNA P-Fl (FRET-1), followed by FRET from DNA P-Fl to EB (FRET-2) takes place. In view of the observed fluorescein or EB emission changes, DNA can be detected in aqueous solution. Because the base mismatch in target DNA inhibits the transition of DNA P-Fl from the stem-loop to duplex structure, single nucleotide mismatch can be clearly detected.     

Tetrahydrofuran Activates Fluorescence Resonant Energy Transfer from a Cationic Conjugated Polyelectrolyte to Fluorescein‐Labeled DNA in Aqueous Media

A cationic water‐soluble conjugated polyelectrolyte, poly[9,9‐bis(6′′‐(N,N,N‐trimethylammonium)hexyl)fluorene‐co‐alt‐2,5‐bis(6′‐(N,N,N‐trimethylammonium)hexyloxyphenylene) tetrabromide], was synthesized. Fluorescence resonant energy transfer (FRET) experiments between the polymer and fluorescein‐labeled single‐stranded DNA (ssDNA‐Fl) were conducted in aqueous buffer and THF/buffer mixtures. Weak fluorescence emission in aqueous buffer was observed upon excitation of the polymer, whereas addition of THF turned on the fluorescence. Fluorescence self‐quenching of ssDNA‐Fl in the ssDNA‐Fl/polymer complexes as well as electron transfer from the polymer to fluorescein may account for the low fluorescence emission in buffer. The improved sensitization of fluorescence by the polymer observed in THF/buffer could be attributed to the weaker binding between the polymer and ssDNA‐Fl and a decrease in dielectric constant of the solvent mixture, which disfavors electron transfer. THF‐assisted signal sensitization was also observed for the polymer and fluorescein‐labeled double‐stranded DNA (dsDNA‐Fl). These results indicate that the use of cosolvent provides a strategy to improve the detection sensitivity for biosensors based on the optical amplification provided by conjugated polymers.     

Solvent-assisted optical modulation of FRET-induced fluorescence for efficient conjugated polymer-based DNA detection

The solvent effects were studied in fluorescence resonance energy transfer (FRET) from a cationic polyfluorene copolymer (FHQ, FPQ) to a fluorescein (Fl)-labelled oligonucleotide (ssDNA-Fl). Upon addition of dimethyl sulfoxide (DMSO), the optical properties of polymers and the probe dye were substantially modified and the FRET-induced PL signal was enhanced 3.8-37 times, relative to that in phosphate buffer solution (PBS). The hydrophobic interaction between polymers and ssDNA-Fl is expected to decrease in the presence of DMSO, which induces the weaker polymer/ssDNA-Fl complexation with longer intermolecular donor-acceptor separation and perturbs the competition between the FRET and PL quenching processes such as photo-induced charge transfer. The gradual decrease in Fl PL quenching with increasing the DMSO content was investigated by measuring the Stern-Volmer quenching constants (3.3-4.2 × 10(6) M(-1) in PBS, 0.56-1.1 × 10(6) M(-1) in 80 vol% DMSO) and PL lifetime of the excited Fl* in polymer/ssDNA-Fl (600 ps in PBS and 2120 ps in 80 vol% DMSO for FHQ/ssDNA-Fl) in PBS/DMSO mixtures. The substantially reduced PL quenching would amplify the resulting FRET Fl signal. The signal amplification in real DNA detection was also demonstrated with fluorescein-labelled PNA (probe PNA) in the presence of a complementary target DNA and noncomplementary DNA in aqueous DMSO solutions. This approach suggests a simple way of modifying the fine-structure of polymer/ssDNA-Fl and improving the detection sensitivity in conjugated polymer-based FRET bioassays.     

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.     

Combinatorial Energy Transfer between an End‐Capped Conjugated Polyelectrolyte and Chromophore‐Labeled PNA for Strand‐Specific DNA Detection

We report a macromolecular end‐capping approach to improve the detection sensitivity of cationic conjugated polymer (CCP) based DNA detection. A phenylethynyl anthracene (PEA) end‐capped cationic polyfluorene (PF) derivative ( P1 ) is synthesized via Suzuki coupling. Due to efficient fluorescence resonance energy transfer (FRET) from the polymer backbone to the end‐capper PEA units, the polymer ( P1 ) fluorescence is dominated by the emission from PEA even in dilute aqueous solution. P1 emission has a better spectral overlap with fluorescein (Fl) absorption compared to that for uncapped PF ( P2 ). In addition, the intra and intermolecular energy transfer for P1 is more efficient in the presence of DNA due to complexation‐induced polymer aggregation. These impart a combinatorial FRET between P1 and an Fl‐labeled probe which is more efficient than that between P2 and the same probe. P1 thus offers a better DNA detection sensitivity relative to P2 and opens up new opportunities to improve the performance of CCP based biosensors involving FRET.

     

Quadruplex-to-duplex transition of G-rich oligonucleotides probed by cationic water-soluble conjugated polyelectrolytes

G-quartet DNA converts to duplex form in the presence of its complementary strand. This conformational change can be detected in real time by a homogeneous assay method based on the signal amplification of conjugated polyelectrolytes and the specific interaction of intercalating dyes with double-stranded DNA (dsDNA). The probe solution contains a cationic, conjugated polymer (CCP), G-quadruplex labeled with a fluorescein at the 5'-terminus (G-quadruplex-Fl), and ethidium bromide (EB). The addition of a complementary target results in the transition from G-quadruplex to duplex (dsDNA-Fl) and EB intercalation within the duplex structure. Excitation of the CCP leads to energy transfer from CCP to dsDNA-Fl (FRET-1) and then energy transfer from dsDNA-Fl to EB (FRET-2). Increasing the number of mismatched bases discourages dsDNA formation, which is detected in the assay.     

Fluorescence measurements of duplex DNA oligomers under conditions conducive for forming M-DNA (a metal-DNA complex)

M-DNA (a metal complex of DNA with millimolar concentrations of Zn2+, Co2+, or Ni2+ and basic pH) has been proposed to undergo electron transfer over long distances along the helix and has generated interest as a potential building block for nanoelectronics. We show that DNA aggregates form under solvent conditions favorable for M-DNA (millimolar zinc and pH = 8.6) by fluorescence correlation spectroscopy. We have performed steady-state F?rster resonance energy transfer (FRET) experiments with DNA oligomers conjugated with 6-carboxyfluorescein and tetramethylrhodamine to the opposite ends of double-stranded DNA (dsDNA) molecules. Enhanced acceptor emission is observed for distances larger than expected for identical DNA molecules with no zinc. To avoid intermolecular FRET, the fluorescently labeled dsDNA is diluted with a 100-fold excess of unlabeled dsDNA. The intramolecular FRET efficiency increases 25-fold for a 30-mer doubly labeled duplex DNA molecule upon addition of millimolar concentrations of zinc ions. Without zinc, this oligomer has less than 1% FRET efficiency. This dramatic increase in the FRET efficiency points to either significant changes in the F?rster radius or fraying of the ends of the DNA helices. The latter hypothesis is supported by our experiments with a 9-mer that show dissociation of the duplex by zinc ions.     

A DNA assay protocol in a lab-on-valve meso-fluidic system with detection by laser-induced fluorescence

An automatic protocol for in-situ assay of dsDNA is presented by employing a micro-sequential injection lab-on-valve meso-fluidic system, which facilitates precise fluidic handling at the 0.1-10 microl level. Sub-nano-liter to a few micro-liters of DNA sample and ethidium bromide (EB) solutions were introduced into the meso-fluidic system, where EB binding onto DNA takes place and an intercalated DNA-EB adduct was formed, which was afterwards excited in the flow cell of the LOV by a 473 nm laser beam, and the emitted fluorescence was monitored in-situvia optical fibers. The experimental variables, i.e., pH of the buffer solution, the concentration and volume of EB solution, the reaction time and the fluid flow rates, were investigated. By loading 600 nl sample and 1.0 microl EB solution, a linear calibration graph was obtained within 0.03-3.0 microg ml(-1)(dsDNA), and a detection limit (3sigma) of 0.009 microg ml(-1) was achieved, along with a sampling frequency of 60 h(-1) and a precision of 1.9% at the 1.0 microg ml(-1) level. The detection limit was further improved to 0.006 microg ml(-1) by increasing the sample volume to 2.0 microl. Plasmid DNA in E. Coli extraction and lambda-DNA/Hind III in four synthetic samples were assayed by using this procedure. For the plasmid DNA, a good agreement with the documented UV method was obtained, while spiking recoveries for the synthetic samples were 95.6-103.4%.     

CO2/NIR light dual-controlled nanoparticles for dsDNA unzipping

Both of carbon dioxide(CO2)and near-infrared(NIR)light as triggers for non-invasive remotely control are attracting wide attentions due to their good biocompatibility and easy operation.Here,CO2/NIR light dual controlled nanoparticles are proposed to remotely regulate the unzipping of dsDNA by using imidazole functionalized conjugated polymer nanoparticles(imidazole-CPNs).The dsDNA successfully coats on the shell of imidazole-CPNs to form imidazole-CPNs/dsDNA assembly due to intensively electrostatic interaction triggered by CO2.Furthermore,the unzipping process of dsDNA is remotely controlled by NIR light based on the photothermal effect,and it can be readily monitored by the fluorescence intensity of ethidium bromide(EB)and CD spectra of dsDNA.Thus,dual stimulation responsive imidazole-CPNs effectively control dsDNA unzipping under CO2 stimulus and NIR light,promising a new direction in the biological applications of DNA,such as the treatments of diseases caused by gene duplication abnormality.     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Water‐soluble dendritic‐conjugated polyfluorenes: Synthesis,characterization, and interactions with DNA

Three generation of Boc‐protected dendritic‐conjugated polyfluorenes ( Boc‐PFP‐G_0‐2 ) were synthesized by Suzuki coupling 1,4‐phenyldiboronic ester with dendritic monomers that were synthesized through generation‐by‐generation approach. The gel permeation chromatography (GPC) analyses showed that the weight‐average molecular weight (M_w) of Boc‐PFP‐G_0‐2 was in the range of 11,400–20,400 Da with the polydispersity index (PDI) in the range of 1.32–1.96. Treatment of Boc‐protected polymers with 6 M HCl in dioxane yielded cationic dendritic‐conjugated polyfluorenes ( PFP‐G_0‐2 ). They were soluble in common polar solvents such as DMSO, DMF, and water with absorption maxima between 345 and 379 nm. The solutions of PFP‐G_0‐2 in water were highly fluorescent with emission maxima between 416 and 425 nm. Because higher generation dendrons could prevent the formation of π‐stacking aggregates of backbones of conjugated polymer, the fluorescence quantum efficiencies (QEs) of PFP‐G_0‐2 enhance as the dendritic generation grew. The interactions between 25 mer double‐stranded DNA (dsDNA) and PFP‐G_0‐2 were studied using ethidium bromide (EB) as fluorescent probe. The electrostatic bindings of PFP‐G_0‐2 with dsDNA/EB complex result in displacement of EB from DNA double helix to the solution accompanying by a quenching of EB fluorescence. The PFP‐G₂ with highest generation of dendritic side chains possessed a highest charge density and could form most stable complex with dsDNA. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7462–7472, 2008     

Shape-specific detection based on fluorescence resonance energy transfer using a flexible water-soluble conjugated polymer

We present the detection of the shape-specific conformation of DNA based on the fluorescence resonance energy transfer (FRET) by using a novel flexible water-soluble cationic conjugated polymer (CCP). The flexible backbone of CCP has more conformational freedom with the potential to be responsive to analyte shape by electrostatic interaction between flexible CCP and negatively charged DNA. The analyte shape dependent recognition is accomplished by structural changes that compressed or extended the flexible CCP. The morphology-dependent spectral properties of the novel flexible polymer related to the analyte shapes are investigated in detail, where two types of chromophores, referred to as "isolated" segment and "packed" segment aggregates, within the flexible polymer are identified by means of ensemble and single molecule measurements upon binding with different geometric DNA. The change in fluorescence intensity upon binding with shape-specific DNA without obvious color shifts makes this novel flexible polymer a suitable CCP donor for FRET measurements. The results provide insights for understanding the spectral properties of flexible water-soluble CCP and CCP/DNA interaction related to the geometry of target analyte.     

Solvent- and pH-induced self-assembly of cationic meta-linked poly(phenylene ethynylene): effects of helix formation on amplified fluorescence quenching and Förster resonance energy transfer

We reported here the synthesis and characterization of a novel water-soluble, meta-linked poly(phenylene ethynylene) (m-PPE-NEt(2)Me(+)) featuring quaternized side groups. We studied the solvent-induced self-assembly of m-PPE-NEt(2)Me(+) in MeOH/H(2)O solvent mixtures by using UV-vis absorption and fluorescence spectroscopies. The results showed that the polymer folded into a helical conformation and that the extent of helical folding increased with the volume % water in the solvent. This cationic polymer also exhibited unique pH-induced helix formation, which was attributed to the partial neutralization of quaternized side groups at high pH and the meta-links in the main chain of the polymer. Studies on the fluorescence quenching of m-PPE-NEt(2)Me(+) by anthraquinone-2,6-disulfonate (AQS) and Fe(CN)(6)(4-), two small-molecule anionic quenchers with different typical structures, revealed more efficient quenching of helical conformation by AQS than by Fe(CN)(6)(4-). We proposed that the two quenchers most likely interacted with the polymer helix in two different modes; that was, AQS featuring large planar aromatic ring could intercalate within adjacent π-stacked phenylene ethynylene units in the polymer helix, whereas Fe(CN)(6)(4-) mainly bound to the periphery of polymer helix through ion-pair formation. Finally, the results of FRET from the helical polymer to the fluorescein (C*)-labeled polyanions, ssDNA-C* (ssDNA: single-stranded DNA) and dsDNA-C* (dsDNA: double-stranded DNA) also suggested two different modes of interactions. As compared with the FRET to dsDNA-C*, the FRET to ssDNA-C* was slightly more efficient, which was believed to arise from the additional binding of ssDNA-C* with the polymer via intercalation of its exposed hydrophobic bases into the π stack of adjacent phenylene ethynylene units in the polymer helix.     

Optimization of interactions between a cationic conjugated polymer and chromophore-labeled DNA for optical amplification of fluorescent sensors

Cationic conjugated polymers (CCPs) have been widely utilized as signal amplifiers in biosensors to improve the detection sensitivity through fluorescence resonance energy transfer (FRET) from CCPs to dye-labeled probes or targets. This paper investigates the effect of sodium dodecyl sulfate (SDS) on energy transfer between a cationic polyfluoreneethynylene copolymer (P1) and Texas Red labeled single-stranded DNA (ssDNA-TR). The presence of SDS in solution affects both the optical properties of P1 and TR emission within P1/ssDNA-TR complexes, which provides basic information on the role of SDS in FRET between P1 and ssDNA-TR. Although the quantum yield of P1 decreases in the presence of low concentrations of SDS, the presence of SDS reduces TR fluorescence quenching within P1/ssDNA-TR complexes and increases the number of optically active polymer repeat units within the proximity of TR, which are beneficial to P1-sensitized TR emission. In the absence of SDS, FRET from P1 to ssDNA-TR provides a 2.6-fold enhancement in TR emission intensity as compared to that upon direct excitation of TR at 595 nm. At the optimum SDS concentration (5 microM), P1-sensitized TR signal output increases to 11.3-fold relative to direct excitation of TR. This study highlights the importance of modulation of the CCP/ssDNA-dye interaction in improving the signal output of dye-labeled DNA by CCP through FRET.     

A Micellar Complex of a Conjugated Polyelectrolyte for Efficient FRET to Dye‐Labeled DNA

A polymer–surfactant micellar complex has been studied as a fluorescence resonance energy transfer (FRET) donor to fluorescein‐labeled DNA (ssDNA‐Fl). In water, the molar absorptivity and fluorescence quantum efficiency of cationic poly(fluorene‐co‐phenylene) (c‐PFP) are substantially increased in the presence of non‐ionic surfactants. A TEM microscopic study shows the formation of a nanowire micellar complex of c‐PFP and the surfactants. About a 400% enhancement of the FRET signal is measured in c‐PFP/ssDNA‐Fl with Brij 30, relative to that without surfactants. The signal amplification is successfully modulated using different types of non‐ionic surfactants which perturb the complexation, fine‐structure of the complex (i.e., donor‐acceptor separation), and the resulting energy transfer process.

     

Fluorescence study of DNA binding and bending by EcoRI DNA methyltransferase

We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .     

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.     

Detection of DNA based on fluorescence resonance energy transfer of polyelectrolyte-protected CdTe quantum dots as energy donors

The approach for DNA detection was established by using a fluorescence resonance energy transfer (FRET) system, in which the energy donor was poly-diallyldimethylammonium chloride-protected quantum dots and the energy receptor was ethidium bromide (EB) inserting into the double stranded DNA. The concentration of the probe DNA, EB and NaCl was optimized. Under the optimized conditions, the FRET system has a stable signal and good reproducibility. The linear range is 7.7-61.6 nM with the correlation coefficient of 0.998 and the limit of detection is 7.7 nM. This method is simple and sensitive, and makes the label-free DNA detection come true.