DNA Hybridization Detection with Water-Soluble Conjugated Polymers and Chromophore-Labeled Single-Stranded DNA

Gaylord, Brent S.; Heeger, A.J.; Bazan, Guillermo C.

doi:10.1021/ja027152+

Cited by 431 publications

(325 citation statements)

References 37 publications

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“…Experimental separation distances were reported for time-resolved spectroscopy measurements and provided values within the estimated R o of 37.2 Å. These measurements confirm that for short, oligomeric DNA targets, the rate of transfer is faster than polymer emission and, thus, effective energy transfer from donor to acceptor is observed (17,18). Because the energy transfer process shows strong distance dependence, it is important to consider the relative length scales of the target DNA and cationic CP.…”

Section: Resultssupporting

confidence: 52%

“…Recently, a collective response method for DNA detection that provides amplified levels of hybridization probe sensitivity was reported (17,18). The assay introduced the use of cationic water-soluble conjugated polymers as light-harvesting additions to standard, singly modified hybridization probes [peptide nucleic acid (PNA) or DNA] by means of FRET.…”

mentioning

confidence: 99%

“…Noncomplementary DNA targets will not form the negatively charged duplex and little attraction is expected between the neutral PNA-FL and cationic CP antenna. Overall, the system provides elevated fluorescent signals and eliminates the need for dual probe modification while maintaining high sequence specificity (17,18).…”

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confidence: 99%

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SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

Gaylord

Massie

Feinstein

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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166

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A strategy employing a combination of peptide nucleic acid (PNA) probes, an optically amplifying conjugated polymer (CP), and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, and sensitive manner. The recognition is accomplished by sequence-specific hybridization between the uncharged, fluorescein-labeled PNA probe and the DNA sequence of interest. After subsequent treatment with S1 nuclease, the cationic water soluble CP electrostatically associates with the remaining anionic PNA͞ DNA complex, leading to sensitized emission of the labeled PNA probe via FRET from the CP. The generation of fluorescent signal is controlled by strand-specific electrostatic interactions and is governed by the complementarity of the probe͞target pair. To assess the method, we compared the ability of the sensor system to detect normal, wild-type human DNA sequences, and those sequences containing a single base mutation. Specifically, we examined a PNA probe complementary to a region of the gene encoding the microtubule associated protein tau. The probe sequence covers a known point mutation implicated in a dominant neurodegenerative dementia known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), which has clinical and molecular similarities to Alzheimer's disease. By using an appropriate PNA probe, the conjugated polymer poly[(9,9-bis(6 -N,N,Ntrimethylammoniumhexylbromide)fluorene)-co-phenylene] and S1 nuclease, unambiguous FRET signaling is achieved for the wildtype DNA and not the mutant sequence harboring the SNP. Distance relationships in the CP͞PNA assay are also discussed to highlight constraints and demonstrate improvements within the system. biosensor ͉ energy transfer ͉ hybridization probes ͉ polyelectrolyte ͉ fluorescence

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Section: Resultssupporting

confidence: 52%

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confidence: 99%

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SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

Gaylord

Massie

Feinstein

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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166

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“…By taking advantage of the intrinsic fluorescence signalamplification properties of CPEs, several groups have recently developed sensitive assays for biologically relevant targets including proteins (1,8,9), DNA (3,(10)(11)(12), glycopeptides (13), and carbohydrates (14). Typical CPE-based fluorescence assays allow detection of the target analytes in the nanomolar concentration range (1,3,10), and, in a few cases, detection limits in the picomolar range have been reported (11).…”

mentioning

confidence: 99%

“…Typical CPE-based fluorescence assays allow detection of the target analytes in the nanomolar concentration range (1,3,10), and, in a few cases, detection limits in the picomolar range have been reported (11). The CPE-based assays share the common features of being relatively easy to implement and giving a rapid response.…”

mentioning

confidence: 99%

Amplified fluorescence sensing of protease activity with conjugated polyelectrolytes

Pinto

Schanze

2004

Proc. Natl. Acad. Sci. U.S.A.

321

222

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Fluorescent conjugated polyelectrolytes with pendant ionic sulfonate and carboxylate groups are used to sense protease activity. Inclusion of the fluorescent conjugated polyelectrolyte into the assay scheme leads to amplification of the sensory response. The sensing mechanism relies on an electrostatic interaction between the conjugated polyelectrolyte and a peptide substrate that is labeled with a fluorescence quencher. Enzyme activity and hydrolysis kinetics are measured in real time by using fluorescence spectroscopy. Two approaches are presented. In the first approach, a fluorescence turn-on sensor was developed that is based on the use of p-nitroanilide-labeled peptide substrates. In this system enzyme-catalyzed peptide hydrolysis is signaled by an increase in the fluorescence from the conjugated polyelectrolyte. The turn-on system was used to sense peptidase and thrombin activity when the concentrations of the enzyme and substrate are in the nanomolar regime. Kinetic parameters were recovered from real-time assays. In the second approach, a fluorescence turn-off sensor was developed that relies on a peptide-derivatized rhodamine substrate. In the turn-off system enzyme-catalyzed peptide hydrolysis is signaled by a decrease in the fluorescence intensity of the conjugated polyelectrolyte.W ater-soluble, fluorescent -conjugated polyelectrolytes (CPEs) afford a unique platform for the development of highly sensitive fluorescence-based sensors for biological targets (1-4). Signal amplification in CPEs results from a combination of factors, including delocalization and rapid transport of the singlet exciton along the -conjugated backbone, along with the propensity of oppositely charged excited-state quenchers to ion-pair with the ionic groups attached to the CPE chains (1, 5-7). In effect, the CPE acts as a highly effective ''antenna'' that channels excitation energy to a quencher trap site that is ion-paired to the CPE. Chemo-optical signal amplification as high as 1,000-fold has been achieved with CPEs, as assessed by the ability to detect molecular fluorescence quenchers (1, 5).By taking advantage of the intrinsic fluorescence signalamplification properties of CPEs, several groups have recently developed sensitive assays for biologically relevant targets including proteins (1,8,9), DNA (3, 10-12), glycopeptides (13), and carbohydrates (14). Typical CPE-based fluorescence assays allow detection of the target analytes in the nanomolar concentration range (1, 3, 10), and, in a few cases, detection limits in the picomolar range have been reported (11). The CPE-based assays share the common features of being relatively easy to implement and giving a rapid response. Moreover, because the response is detected by using fluorescence, the systems can be adopted to a format that is compatible with f luorescence-based highthroughput screening (HTS) assays (15).In most cases, CPE-based bioassays take advantage of a competition between a nonspecific ion-pairing of an ionic quencher moiety to the CPE chain and the highly sp...

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Colorimetric Sensor Design

2011

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DNA Hybridization Detection with Water-Soluble Conjugated Polymers and Chromophore-Labeled Single-Stranded DNA

Cited by 431 publications

References 37 publications

SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification

Amplified fluorescence sensing of protease activity with conjugated polyelectrolytes

Colorimetric Sensor Design

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