Size‐Specific Interactions Between Single‐ and Double‐Stranded Oligonucleotides and Cationic Water‐Soluble Oligofluorenes

521

348

We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly "universal" biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise "folded" DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient-picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.biosensor | aptamer | visual detection | thrombin detection | cocaine detection G old nanoparticle colorimetric biosensors have seen significant applications in diagnostics, environmental monitoring, and antibioterrorism supporting unaided, visual readout (1-12). Commonly, the relevant nanoparticles are covalently modified with either a probe DNA or an aptamer such that hybridization (13-16) or aptamer-target interactions (17-27), for example the scanometric method developed by Mirkin (25), which is a very sensitive and specific tool, crosslink them, inducing aggregation. The second broad approach utilizes unmodified nanoparticles. (28-30) These two approaches, however, suffer from timeconsuming (20-40 h of assembly) and relatively poor (low nanomolar) detection limits, respectively. Here, a unique, colorimetric sensing strategy employing a simple but selective combination of a single-stranded DNA probe, a positively charged, water-soluble conjugated polyelectrolyte, and unmodified gold nanoparticles is demonstrated. The universality of this method allows detection of a broad range of targets, including nucleic acid (DNA) sequences, proteins, small molecules, and ino rganic ions. Our approach is rapid (turnaround time is 5-10 min) and sensitive (picomolar concentrations of target DNA are readily detected with the naked eye, even in complex sample matrices like blood serum). Hence, an operator with minimum scientific overhead can easily employ this technique.Generally, the gold nanoparticle applications typically rely on a quantitative coupling between target recognition and the aggregation of the nanoparticles, which, in turn, leads to a dramatic change in the photonic properties-and thus the color-of the nanoparticle solution. This colorimetric "readout" avoids the relative complexity inherent...

mentioning

confidence: 99%

Colorimetric detection of DNA, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes

Xia

Zuo

Yang

et al. 2010

521

348

“…Water-soluble conjugated polymers (conjugated polyelectrolytes) are obtained by terminating the solubilizing side groups of traditional conjugated chains, e.g., poly(phenylene vinylenes) or polyf luorenes, with charged moieties (8,9,11,12). Positively charged (cationic) conjugated polymers (CCPs) are particularly useful because biopolymers such as DNA and RNA are negatively charged.…”

mentioning

confidence: 99%

Time-resolved energy transfer in DNA sequence detection using water-soluble conjugated polymers: The role of electrostatic and hydrophobic interactions

Gaylord

Wang

et al. 2004

107

We have investigated the energy transfer processes in DNA sequence detection by using cationic conjugated polymers and peptide nucleic acid (PNA) probes with ultrafast pump-dump-emission spectroscopy. Pump-dump-emission spectroscopy provides femtosecond temporal resolution and high sensitivity and avoids interference from the solvent response. The energy transfer from donor (the conjugated polymer) to acceptor (a fluorescent molecule attached to a PNA terminus) has been time resolved. The results indicate that both electrostatic and hydrophobic interactions contribute to the formation of cationic conjugated polymers͞PNA-C͞ DNA complexes. The two interactions result in two different binding conformations. This picture is supported by the average donor-acceptor separations as estimated from time-resolved and steady-state measurements. Electrostatic interactions dominate at low concentrations and in mixed solvents. C onjugated polymers are novel materials with useful optical and electronic properties (1). Although structural disorder causes the effective localization length (conjugation length) to be significantly shorter than the actual chain length, an excitation can migrate along the chain before it is quenched via electron transfer to a nearby quencher (2-5) or before the excitation energy is transferred to a nearby acceptor (6 -10). Thus, conjugated polymers function as light-harvesting materials and thereby exhibit greatly enhanced quenching efficiencies via electron transfer (2-5) and optical amplification via Förster resonance energy transfer (FRET) (6 -10). Because of these exceptional properties, conjugated polymers offer potential for use in detecting biological and chemical target molecules with high sensitivity (2, 7-10).To develop homogenenous biosensors, water solubility is essential. Water-soluble conjugated polymers (conjugated polyelectrolytes) are obtained by terminating the solubilizing side groups of traditional conjugated chains, e.g., poly(phenylene vinylenes) or polyf luorenes, with charged moieties (8,9,11,12). Positively charged (cationic) conjugated polymers (CCPs) are particularly useful because biopolymers such as DNA and RNA are negatively charged. When these negatively charged biopolymers are labeled with f luorescent molecules, electrostatic interactions bring the f luorescent labels sufficiently close to the CCP to enable fast and efficient FRET.The long-range excitation energy transfer from donor to acceptor via the dipole-dipole interaction was initially described by Förster (6). The rate of energy transfer for donor and acceptor separated by a distance r DA is given by (13):D is the lifetime of donor in the absence of acceptor, Q D is the quantum yield of the donor in the absence of acceptor, N is Avogadro's number, n is the refractive index of the medium (typically assumed to be 1.4 for biomolecules in aqueous solution), and 2 is a factor describing the relative orientation of the transition dipoles of the donor and acceptor. The integral, J(), expresses the degree of spectral o...

“…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. Based on the molecular weight of the polymer (M w Ϸ 13,000 g͞mole, Ϸ13 Å per repeat unit), it is evident that the CP is longer than the oligonucleotide (15-28 base) target sequences tested previously (27)(28)(29)(30)(31)44).…”

Section: Resultsmentioning

confidence: 99%

“…This collective behavior gives rise to optical enhancement or amplification if the lowenergy site comprises a reporter fluorophore. The water solubility of these macromolecules is achieved by attaching ionic side chains pendent to the otherwise hydrophobic main chain, whereas the spectral properties are tuned by altering the actual conjugated backbone structure (26)(27)(28)(29)(30)(31).…”

mentioning

confidence: 99%

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

Gaylord

Massie

Feinstein

et al. 2004

165

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