Fluorescence images of DNA-bound YOYO between coupled silver particles

Oligonucleotide-bound silver particles were coupled through hybridization with target complementary oligonucleotides. YOYO molecules were intercalated into DNA duplexes bound between the coupled metal particles. Fluorescence images of YOYO molecules were monitored by scanning confocal microscopy. Relative to the free single YOYO, the emission brightness of the image was enhanced 80-fold after intercalating the fluorophores into the DNA duplexes between the coupled silver particles. Some images of the labeled metal particle dimers were observed to be dumbbell-shaped, suggesting that the stretching of intercalated YOYO molecules was restricted because of the orientation effect of fluorophores. The shortened lifetime of YOYO molecules between the coupled metal particles indicated that the fluorescence was enhanced via a near-field interaction mechanism between the fluorophore and the metal nanoparticle.     

A model for DNA detection by metal-enhanced fluorescence from immobilized silver nanoparticles on solid substrate

N-(2-Mercaptopropionyl)glycine (tiopronin)-coated silver nanoparticles (average diameter of metal core=5 nm) were prepared by a modified Brust method. Tiopronin ligands were partially displaced by thiolate single-stranded oligonucleotides via ligand exchange. These particles were immobilized onto a solid substrate through hybridization with target oligonucleotides in a layer-by-layer approach. The dye-labeled complementary oligonucleotides were bound to the particle layers through hybridization. Fluorescence intensity was enhanced with a simultaneous increase of plasmon absorbance from accumulated particles. A steady state was shown at the 10th particle layer and then the fluorescence enhancement showed a plateau. This result reveals that increasing the particle layer contributes to fluorescence enhancement. This novel method was used to detect DNA hybridization through both absorbance and emission spectral changes.     

Surface enhanced fluorescence and Raman imaging of Langmuir-Blodgett azopolymer films

The spectroscopic properties and surface-enhanced spectra of Langmuir-Blodgett (LB) films of methacrylic homopolymer (HPDR13) are presented. It is shown that LB film displays strong fluorescence attributed to the spatial restrictions imposed by its structure. The emission is observed in conjunction with photoisomerization, a process clearly demonstrated by the formation of surface-relief gratings in the LB film [C.R. Mendon?a et al., Macromolecules 32 (1999) 1493]. Surface-enhanced Raman scattering (SERS), Surface-enhanced resonance Raman scattering (SERRS) and surface-enhanced fluorescence (SEF) were observed for LB films of HPDR13 deposited onto silver island films. SERS measurements were also carried out on a sample fabricated with one monolayer LB film deposited onto silver islands followed by one overlayer of silver (LB sandwiched between two layers of silver islands). The polymer interacts very weakly with the metal surface (physisorption), and the enhancement effect is determined by the local electric field enhancement. The strong SERS and SERRS signals were suitable for micro-Raman imaging. Line, area mapping and global images of the LB monolayer on silver island are reported. The transfer ratio in the fabrication of the LB suggests a homogeneous coating of the silver islands, thereby the chemical images show the variation of the SERS intensity due to surface enhancement.     

Surface enhanced resonance Raman scattering imaging of Langmuir-Blodgett monolayers of bis (benzimidazo) thioperylene.

The synthesis, spectroscopic characterization and surface-enhanced spectroscopy of a new electro active organic material bis (benzimidazo) thioperylene (Monothio BZP) are reported. Langmuir monolayers of Monothio BZP were successfully formed on water subphase and characterized by the pi-A surface-pressure area isotherm. Langmuir-Blodgett (LB) monomolecular layers of Monothio BZP were fabricated onto glass substrates and onto silver island films for surface-enhanced spectroscopic studies. The results of surface-enhanced resonance Raman scattering (SERRS), SERRS imaging and surface-enhanced fluorescence (SEF) studies for Monothio BZP LB monolayers are reported. Raman imaging (global imaging and point-by-point mapping) of the SERRS signal for a single monomolecular layer on silver islands were obtained using the 514.5 nm laser line. The SERRS imaging permits a visualization of the variation of the SERRS intensity across of the rough metal surface. The SEF was recorded for the excimer emission of aggregates in the LB film. The distance dependence and the enhancement factor of SEF were determined using fatty acid spacing layers. A temperature dependence study of the LB monolayer SERRS and SEF spectra was carried out between -190 degrees and + 200 degrees C confirming the thermal stability of the LB monolayer on silver. The specificity and the sensitivity of SERRS signal on metal island films was probed using mixed LB films with 0.01% molecular ratio of Monothio BZP in Arachidic Acid (AA). The micro-Raman SERRS spectra from ca. 10(-3) attomole of the dye were recorded.     

Surface-enhanced fluorescence and surface-enhanced Raman scattering of push–pull molecules: sulfur-functionalized 4-amino-7-nitrobenzofurazan adsorbed on Ag and Au nanostructured substrates

We investigated the chemisorption of self-assembled monolayers of sulfur-functionalized 4-amino-7-nitrobenzofurazan on gold and silver nanoisland films (NIFs) by means of surface-enhanced fluorescence (SEF) and surface-enhanced Raman scattering (SERS). The ligand is a push–pull molecule, where an intramolecular charge transfer occurs between an electron-donor and an electron-acceptor group, thus exhibiting nonlinear optical properties that are related to both SERS and SEF effects. The presence of different heteroatoms in the molecule ensures the possibility of chemical interaction with both silver and gold substrates. The SERS spectra suggest that furazan is bound to silver via lone pairs of the nitrogen atoms, whereas the ligand is linked to gold via a sulfur atom. Silver NIFs provide more efficient enhancement of both fluorescence and Raman scattering in comparison with gold NIFs. The present SEF and SERS investigation could provide useful information for foreseeing changes in the nonlinear responses of this push–pull molecule.     

Metal-enhanced fluorescent probes based on silver nanoparticles and its application in IgE detection

In this paper, a novel metal plasmon coupled with an aptamer–nucleotide hybridized probe was fabricated and applied for protein detection. The specific aptamer and single-strand oligonucleotide were chemically bound to silver nanoparticles (AgNPs), and Cy5-labeled, complementary single-strand oligonucleotides were hybridized with the particle-bound oligonucleotides. The hybridized DNA duplexes were regarded as rigid rods that separated the fluorophore Cy5 and the surface of AgNPs to reduce the competitive quenching. Using a model system comprising human immunoglobulin E (IgE) as the analyte and goat antihuman IgE as immobilized capture antibody on glass slides, we demonstrate that the detection performance of the synthetic probe was superior to the aptamer-based fluorescent probes. The results showed a good linear correlation for human IgE in the range from 10 ng/ml to 6.25 μg/ml. The detection limit obtained was 1 ng/ml, which was 50 times lower than that using Cy5 oligonucleotide/aptamer hybrid duplex (Probe2) due to the metal-enhanced fluorescence effect. This new strategy opens the possibility for the preparation of high-sensitivity detection probes based on metal nanoparticles.     

Emission control by binary energy transfer processes on oligouridine

Fluorescent oligonucleotides have been designed on which two different energy transfer processes were mounted together: excitonic interaction and FRET. The fluorescence emission of the oligonucleotides was controlled well by the two different energy transfer processes, in response to their hybridization to the complementary RNA both in vitro and in cells.     

The oxime bond formation as an efficient tool for the conjugation of ruthenium complexes to oligonucleotides and peptides

A convenient method for the conjugation of ruthenium complex on oligonucleotides and peptides through chemoselective oxime linkage is reported. Novel Ru(II) complexes sustaining an aminooxy containing ligand were prepared and efficiently coupled with the oligonucleotides and peptides functionalized with the complementary reactive aldehyde group. The method described herein could be a useful tool for preparing a broad range of metal complex-oligonucleotide and peptide conjugates.     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Synthesis and Hybridization Properties of Oligodeoxynucleotides with Long‐Chain Linkers

New oligonucleotides with a long‐chain linker (6,9‐dioxa‐3,12‐diazatetradecane‐1,14‐diyl) in their backbone were synthesized, and their hybridization properties were studied by measurement of their T_m curves and fluorescence spectra. The T_m analyses revealed that these oligonucleotides could bind to their complementary strands despite the presence of the long‐chain linker. We also demonstrated interesting fluorescence properties of oligodeoxynucleotides with an anthracen‐9‐ylmethyl group on one of the two N‐atoms in the long‐chain linker. The fluorescence intensity of these oligonucleotides increased upon their hybridization to the complementary strands and was sensitive to the presence of the mismatch base pairs at a specific position.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 microL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides.     

Enzymatic manipulations of DNA oligonucleotides on microgel: towards development of DNA-microgel bioassays

We demonstrate that DNA oligonucleotides covalently coupled to colloidal microgel can be manipulated by T4 DNA ligase for DNA ligation and by Phi29 DNA polymerase for rolling circle amplification (RCA). We also show that the long single-stranded RCA product can generate intensive fluorescence upon hybridization with complementary fluorescent DNA probe. We believe DNA-microgel conjugates can be explored for the development of DNA based bioassays and biosensors.     

Nanoparticle decorated surfaces with potential use in glycosylation analysis

A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range. The nanoparticles present high concentrations of attachment sites for specific ligands and cause minimal steric hindrance to binding. In the present model study the mannose-binding lectin ConA has been coupled to polystyrene nanoparticles via a poly(ethyleneoxide) linker which protects the protein conformation and activity and prevents unspecific protein adsorption. The ConA-coated particles are accommodated at different spots on the analytical surface via oligonucleotide linkage. This attachment, which relies on the hybridization of complementary oligonucleotides, allows firm fixation of the particles at specific positions. The ConA attached to the particles has retained conformation and activity and binds selectively to a series of different glycoproteins. The results indicate the potential for using a multi-lectin nanoparticle array in glycoprotein mapping.     

Angular-dependent metal-enhanced fluorescence from silver colloid-deposited films: opportunity for angular-ratiometric surface assays

We describe an exciting opportunity for Metal-Enhanced Fluorescence (MEF)-based surface assays using an angular-ratiometric approach to the observed enhanced emission from fluorophores in close proximity to silver colloids deposited on glass substrates. This approach utilizes the radiationless energy transfer (coupling) between the excited states of the fluorophore and the induced surface plasmons of the silver colloids, and the subsequent angular-dependent fluorescence emission from the fluorophore-silver colloid system. Since MEF is related to surface plasmons' ability to scatter light, angular-dependent light scattering from three different silvered surfaces and glass substrates were investigated using two common excitation angles, 45 and 90 degrees . The scattered light from silvered surfaces with a high loading was observed at wider angles on both sides of the glass substrates, while forward scattering (from the back of the glass) was dominant for the silvered surfaces with low loading, as explained by both Mie and Rayleigh theories. When silver colloids were placed between the fluorophore and glass interface, the coupled fluorescence emission through the higher refractive index glass (and in air), increased in an angular-dependent fashion, following closely the angular-dependent light scattering pattern of the silver colloids themselves. Similar observations for fluorescence emission from fluorophores deposited onto glass surfaces alone were made, but at much narrower angles on both sides of the fluorophore-glass interface and were simply explained by Lambert's cosine law. As the loading of silver on glass was increased, the enhanced fluorescence emission was observed at wider angles (towards 0 and 180 degrees ) at both sides of the silvered surfaces. Glass surfaces without silver colloids were used as control samples to demonstrate the benefits of MEF for enhancing fluorescence signatures in an elegant, angular-dependent fashion. Finally, the utility of the angular-dependent MEF phenomenon for intensity-based angular-ratiometric surface assays is demonstrated.     

Enhanced fluorescence images for labeled cells on silver island films

Silver island films (SIFs) were deposited on glass substrates to serve as supports. T-Lymphocytic (PM1) cell lines were labeled by Alexa Fluor 680-dextran conjugates on the membranes or by YOYO in the nuclei. The fluorescence images of the cell lines were recorded in the emission intensity and lifetime using scanning confocal microscopy. The fluorescence signals by the fluorophores bound on the cell membranes were enhanced significantly by SIF supports as compared with those on the glass. In addition to the increase in the intensity, there was a dramatic shortening of the emission lifetime. In contrast to the Alexa Fluor 680 fluorophores on the membranes, the YOYO fluorophores intercalated in the cell nuclei were not influenced significantly by the silver islands. This result can be interpreted by an effect of the distance on coupling between the fluorophores and metal particles: the fluorophores on the cell membranes are localized within, but the fluorophores in the cell nuclei are beyond the region of metal-enhanced fluorescence. Thus, the metal supports can be used to improve the detection sensitivity for target molecules on cell surfaces when they are fluorescently labeled.     

Large‐Area Plasmonic Substrate of Silver‐Coated Iron Oxide Nanorod Arrays for Plasmon‐Enhanced Spectroscopy

One‐dimensional iron oxide materials fabricated on conducting glass substrates and their unique properties make these nanostructures promising candidates for a wide range of applications. Herein, vertically oriented α‐Fe₂O₃ nanorod arrays synthesized under hydrothermal conditions over a large area are described, as an active platform for surface‐enhanced resonance Raman scattering (SERRS) and surface‐enhanced fluorescence (SEF). From scanning electron microscopy images the formation of a homogeneous distribution of vertically oriented rods in a large area is confirmed. For activating the localized surface plasmon resonances, which are responsible for SERRS and SEF, a 6 nm layer of Ag is deposited onto the α‐Fe₂O₃ nanorod arrays by physical vapor deposition to form Ag islands.     

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA. A stop-flow system of 137 μL volume permitted rapid introduction and mixing of each sample. Response times measured were on the order of seconds to minutes. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on the extent of hybridization of complementary oligonucleotides, but actually reduced the response times of sensors to cDNA oligonucleotides. Received: 26 September 2000 / Revised: 24 November 2000 / Accepted: 30 November 2000     

Hybridization of oligonucleotide-modified silver and gold nanoparticles in aqueous dispersions and on gold films

Functionalization of silver and gold nanoparticles by 12mer-thiolated homo-oligonucleotides, SA and ST (containing only adenine or thymine, respectively), and their hybridization and dehybridization in aqueous dispersions have been described. In addition, ST and SA were self-assembled onto gold films and hybridized with their complementary pairs, unlabeled or labeled by gold and silver nanoparticles. The base pairing between DNA strands and the types of oligonucleotides (adenine or thymine) attached to the nanoparticles was detected by Polarization Modulated Fourier Transform Infrared Reflection Absorption Spectroscopy (PM-FTIRRAS).     

Cantilever tip near-field surface-enhanced Raman imaging of tris(bipyridine)ruthenium(II) on silver nanoparticles-coated substrates

The near-field surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) images of tris(bipyridine)ruthenium(II) adsorbed on a silver nanoparticles-coated substrate were obtained with a scanning near-field optical microscope (SNOM, or near-field scanning optical microscopy, NSOM) using a cantilever tip. In comparison with the most widely used fiber tip for SNOM, the cantilever tip has higher optical throughput and better thermal stability, making it more suitable for detecting the extremely low Raman signal in the near-field spectroscopic investigations. Our preliminary results show that the near-field SERS with the higher spatial resolution can provide richer fingerprint information than the far-field SERS. A comparison of the two types of images shows that there are more SERS than SEF hot spots, and the two types of hot spots do not overlap. More surprisingly, the near-field SERS spectra differ from the far-field SERS spectra obtained on the same sample in the band frequency and relative intensities of some major Raman bands, and some IR-active bands were observed with the near-field mode. These results are explained mainly by the electric field gradient effect and heterogeneous polarization character that operate only in the near-field SERS.     

Conformational change induced by metal-ion-binding to DNA containing the artificial 1,2,4-triazole nucleoside

A conformational switch can be induced upon the addition of transition-metal ions to oligonucleotides that contain a row of successive artificial nucleobases flanked by complementary sequences of natural nucleobases, provided that the artificial bases cannot undergo self-pairing via hydrogen bonding but only via the formation of metal-ion-mediated base pairs. Such oligonucleotides adopt a hairpin structure in the absence of transition-metal ions, yet they show a preference for the formation of a regular double helix if the appropriate metal ions are present. We report here our experimental data on the structure of the oligonucleotide d(A7X3T7) (A=adenine, T=thymine, X=1,2,4-triazole) in the absence and presence of silver(I). This study comprising temperature-dependent UV spectroscopy, CD spectroscopy, MALDI-TOF measurements, fluorescence spectroscopy, and dynamic light scattering opens up a new approach to the generation of a large variety of metal-ion sensors with the possibility of fine-tuning their sensing capabilities, depending on the artificial nucleoside that is used.