DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

A s the human genome project has uncovered the full sequence of human genomes and the postgenome technologies have been rapidly developed, DNA hybridization detection has become increasingly important. 1 A variety of DNA detection formats are now available, including both heterogeneous assays with surface-bound array-based probes and homogeneous solution-based detection schemes. 2 A number of those rely on fluorescence signal and, in particular, on fluorescence (or Fö rster) resonant energy transfer (FRET). 3 Both conjugated polymers and semiconductor nanocrystals (colloidal quantum dots, QDs) have been suggested for use as fluorescent entities in DNA detection assays. 4Ϫ12 Assays based on watersoluble cationic conjugated polymers allow one to utilize a simple binding strategy to DNA through electrostatic interactions with ionic side groups and benefit from the lightharvesting properties along the polymer backbone. 4Ϫ8 QDs have been increasingly used as donors in FRET-related studies, including optical DNA diagnostics. 9Ϫ12 QDs are photostable, highly efficient fluorophores with a strong band gap luminescence tunable by size as a result of the quantum confinement effect. 13 FRET in QD/ DNA/organic dye conjugates has been used to study DNA hybridization, 14,15 cleavage, 16 and replication. 17 Most of the reported approaches required covalent conjugation of DNA to the QD surface (e.g., by EDC/NHS coupling reaction), which often decreases the colloidal stability and/or the emission intensity of QDs. 18,19 This limitation can be overcome by the use of a sensing platform relying on the electrostatic interaction of dye-labeled DNA with the oppositely charged QD surface 20 in a similar fashion as in conducting polymer-based detection assays. 4Ϫ8 In this paper, we propose a simultaneous use of conjugated polymers and semiconductor QDs for DNA hybridization detection, which can potentially combine advantages of both light-harvesting and DNA-binding properties of water-soluble polymers with photostability, lightharvesting ability, and FRET donor property of QDs. A blue-emitting conjugated polymer with positively charged side chains electrostatically self-assembles on the negatively charged surface of red-emitting CdTe QDs. The so-formed hybrid complex has a net positive charge and thus attracts negatively charged dye-labeled DNA molecules (Scheme 1). The water-soluble, cationic, conjugated polymer, poly[9,9-bis(3=-((N,N-

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“…15,26 The dual use of both conjugated polymer and QD extinction offers a potential to lower this limit to picomolar range. 4,5,8 …”

Section: Resultsmentioning

confidence: 99%

Cascaded FRET in Conjugated Polymer/Quantum Dot/Dye-Labeled DNA Complexes for DNA Hybridization Detection

et al. 2009

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“…[1][2][3][4][5][6][7][8][9] We show here, however, that unlike molecular beacons, which rely on a rigid, binding-induced conformational change (to segregate a fluorophore-quencher pair), [10][11][12] E-DNA signaling arises due to binding-induced changes in the dynamics of the probe DNA. We do so by demonstrating that hybridization-linked changes in the dynamics of an electrodebound linear (as opposed to stem-loop) probe DNA efficiently support E-DNA signaling.…”

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confidence: 99%

Linear, redox modified DNA probes as electrochemical DNA sensors

2007

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"Linear, redox modified DNA probes as electrochemical DNA sensors" (2007). Rebecca Lai Publications. 2. http://digitalcommons.unl.edu/chemistrylai/2 E-DNA sensors, which consist of a redox-tagged stemloop DNA covalently attached to an interrogating electrode, are the electrochemical equivalents of optical molecular beacons. [1][2][3][4][5][6][7][8][9] We show here, however, that unlike molecular beacons, which rely on a rigid, binding-induced conformational change (to segregate a fluorophore-quencher pair), 10-12 E-DNA signaling arises due to binding-induced changes in the dynamics of the probe DNA. We do so by demonstrating that hybridization-linked changes in the dynamics of an electrodebound linear (as opposed to stem-loop) probe DNA efficiently support E-DNA signaling. That is, whereas a large Faradaic current is observed from a redox-modified, single-stranded DNA probe, this current is reduced upon hybridization to the appropriate target DNA sequence due to changes in the rate with which the terminal redox label collides with the electrode surface ( Figure 1).We have fabricated E-DNA sensors using a 27-base linear probe sequence that, in order to facilitate direct comparison with earlier studies, is directly analogous to a previously characterized stem-loop E-DNA sensor 9,13 save that the five base sequences at the two termini are identical and thus do not form a double stranded stem. In the absence of target, the sensor gives rise to a sharp, well-defined AC voltammetry peak consistent with the ~ -0.26 V (vs. Ag/AgCl) formal potential of the methylene blue redox moiety employed (Figure 2). Upon hybridization to a fully complementary, 17-base target this current is significantly reduced. Furthermore, because the observed signal change arises due to a hybridization-specific change in DNA dynamics (as opposed to the simple adsorption of mass or charge to the sensor surface), we can readily observe this change even when the sensor is challenged with complex, multi-component sample matrices, such as targetdoped blood serum (Figure 2, right). Finally, like the original stem-loop E-DNA architecture, the linear-probe E-DNA sensor is label-free and reusable: a 30 sec wash in room temperature distilled water or (after deployment in blood serum) room temperature detergent solution is enough to regenerate >97% original sensor current (Figure 2).The signaling characteristics of linear probe E-DNA sensors are improved relative to those of the equivalent stem-loop sensor. Whereas a linear probe E-DNA sensor exhibits an 85% signal reduction at a given target concentration (Figure 2), the equivalent stem loop sensor exhibits only 71% signal suppression at this target concentration. 9 We presume this difference Published in Chemical Communications (2007), no. 36, pp. 3768-3770; doi Supplementary information follows the "References," including experimental procedures, controlling probe surface density, sensor equilibration time and specificity, the effect of target length and bulk on signaling, electron transfer rate meas...

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“…20,21 Bazan and coworkers used trimethyl-ammonium containing polyfluorene and demonstrated a concept where specific DNA complementary peptide nucleic acid (PNA) prevents CPE-DNA interaction, thus allowing the use of this polymers optical signal with and without DNA interaction for DNA sensing. 22 Since its introduction, this idea has been expanded for other properties and more complicated materials using the cationic tetra-alkylammonium group. Al Attar, Monkman, Monteserin and others have fine-tuned CPE-DNA interactions by surfactants 23,24 while Inganäs and co-workers detailed properties of zwitterionic CPE hydrogels.…”

Section: Introductionmentioning

confidence: 99%

Conjugated Polyelectrolyte (CPE) Poly{3-[6-(N-methylimidazolium)hexyl]-2,5-thiophene} Complexed with DNA: Relation between Colloidal Level Solution Structure and Chromic Effects

et al. 2014

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We report on the solution structure and colorimetric changes for ~1 % (w/w) aqueous cationic conjugated polyelectrolyte, poly [3-[6-(N-methylimidazolium)hexyl]-2,5-thiophene] bromide (P3ImiHT), mixed with double stranded (anionic) DNA -P3ImiHT(DNA) x , where x is the molar ratio of DNA base pair to polymer repeat unit. Small-angle X-ray scattering (SAXS) indicate that P3ImiHT forms loose arrays of rodlike chains involving electrostatic repulsion. SAXS and transmission electron microscopy indicate that the DNA addition reduces this repulsion and leads to networks of ~10 nm sized aggregates  

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DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

Cited by 584 publications

References 31 publications

Cascaded FRET in Conjugated Polymer/Quantum Dot/Dye-Labeled DNA Complexes for DNA Hybridization Detection

Cascaded FRET in Conjugated Polymer/Quantum Dot/Dye-Labeled DNA Complexes for DNA Hybridization Detection

Linear, redox modified DNA probes as electrochemical DNA sensors

Conjugated Polyelectrolyte (CPE) Poly{3-[6-(N-methylimidazolium)hexyl]-2,5-thiophene} Complexed with DNA: Relation between Colloidal Level Solution Structure and Chromic Effects

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