The photoluminescence (PL) properties of quantum dots (QD) are of significant interest in the development of new
methods for bioanalysis. Multiplexed solid-phase nucleic acid hybridization assays that use immobilized QDs as donors
in fluorescence resonance energy transfer (FRET) are one such example, and offer several unique advantages over other
methods. In this work, new interfacial chemistry is described for the immobilization of red-emitting CdSe/ZnS QDs on
glass beads for use in hybridization assays. The beads were chemically modified with a dithiolate surface ligand and the
QDs immobilized via self-assembly. Further derivatization of the QDs with dithiolate-terminated probe oligonucleotides
enabled a hybridization assay that could detect unlabeled target down to nanomolar levels with discrimination of single
base-pair mismatches. The use of beads as an immobilization platform afforded shorter analysis times and superior
reusability compared to previous studies using optical fibers. Hybridization between probe, target, and Alexa Fluor 647
(A647) labeled reporter oligonucleotides in a sandwich format generated a spectroscopic signal by introducing the
proximity needed for FRET between the QDs and A647. The results indicate clear directions for the optimization of
solid-phase hybridization assays, and are important for the future development of true multiplexed biosensors based on
QDs and FRET.
Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical
multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the
development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green- or red-emitting
QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently
conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe
conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in
microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or
Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the
detection of picomolar quantities of target. The simultaneous immobilization of green- and red-emitting QDs at different
ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.
Quantum dots (QDs) have been widely adopted as integrated components of bioassays and biosensors. In particular,
solid phase nucleic acid hybridization assays have been demonstrated to have several advantages and permit the
detection of up to four DNA targets simultaneously using fluorescence resonance energy transfer (FRET). This work
explores the potential for miniaturization of a solid-phase nucleic acid hybridization assay using QDs and FRET on a
microfluidics platform. A method was developed for the immobilization of Streptavidin coated QDs and the preparation
of QD-probe oligonucleotide conjugates within microfluidic channels using electrokinetic delivery. Proof-of-concept
was demonstrated for the selective detection of target DNA using FRET-sensitized emission from a Cy3 acceptor paired
with a green emitting QD donor. The microfluidic platform offered the advantages of smaller sample volumes, nearly
undetectable non-specific adsorption, and hybridization within minutes. This work is an important first step toward the
development of biochips that enable the multiplexed detection of nucleic acid targets.
The optical properties and significant surface area of CdSe/ZnS QDs make such nanoparticles an interesting platform for
the preparation of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Interactions between
QDs and oligonucleotides affect biosensor performance and are not fully understood. Ensemble data obtained via FRET
experiments indicated that, on average, 4-5 added oligonucleotides saturated the surface of green emitting QDs. An
increase in the number of oligonucleotides per QD appeared to cause the oligonucleotides to transition from collapsed to
upright conformations. Since bulk averaging hides details of such processes, methods must be developed and materials
identified for studying QD-oligonucleotide conjugates at the single molecule level. Single QDs have been immobilized
and fluorescence intensity trajectories measured. High count rates and good photostability were achieved using carboxyl
polymer-coated QDs. Modeling of FRET efficiency based on the dimensions of QDs and oligonucleotides indicated that
transitions between collapsed and upright conformations can be accurately measured based on changes in QD
fluorescence lifetime. The ultimate goal of this work is to elucidate QD-oligonucleotide dynamics for better design and
optimization of nucleic acid biosensors based on QDs.
Interest in porous silicon (PS) has increased dramatically over the past two decades due to aspects such as
photoluminescence due to quantum confinement, large surface area, and micro/nanoscale architecture. In this work,
<111> p-type silicon wafers have been electrochemically etched with ethanolic solutions of hydrofluoric acid. Discrete
surface domains showing luminescence were observed. The domains were typically many tens of micrometers in size
and had a height of about 6-8 μm. Interestingly, central round wells of 10-30 μm diameter were observed to form within
domains. Investigation of luminescence in depth profile of the wells was done using confocal fluorescence microscopy,
and the results indicated that the domains were fully porous and luminescent throughout the entire depth. Spectrally, the
peak fluorescence emission was in the range of 550-750 nm and the spectra had an average FWHM equal to about 150
nm. Covalent attachment of organic monolayers to the porous silicon surfaces was done to try and passivate against
oxidation, and also to explore the possibilities of bioconjugation and tuning of the photoluminescence wavelength. A
reaction of hydrogen terminated silicon with ω-undecylenyl alcohol was done using irradiation by a UV source, and
successful derivatization was confirmed with IR spectroscopy. Bulk electrochemical etching of silicon provided a
method to generate distributed luminescent structures suitable for compartmentalization of reactions within wells of
micrometer dimensions without the use of spatially resolved fabrication methodologies such as epitaxial deposition,
lithography, or ion beam technologies.
Quantum dots (QDs) are nanostructures that are highly attractive to optical biosensing. We have developed a nucleic acid biosensing strategy based on the use of quantum dots as energy donors in FRET. One of the challenges in such an approach is avoiding the non-specific adsorption of oligonucleotides. In this report, we describe our efforts to develop poly(ethylene glycol) (PEG)-based hydrophilic surface chemistry and hexanethiol based hydrophobic surface chemistry to alleviate non-specific adsorption. With respect to the former, it was found that the PEG surface chemistry strongly quenched the band-edge luminescence of CdSe/ZnS QDs and yielded significant band-gap luminescence. Furthermore, the PEG chemistry proved ineffective in preventing adsorption. With respect to hexanethiol capped CdSe/ZnS QDs, it was found that good QD luminescence was retained in organic solvent but was quenched in aqueous solution. The use of hydrophobic hexanethiol QDs in aqueous solution required the immobilization of QDs. To achieve this, we used thiol modified biotin and avidin coated fused silica optical fibers. Despite the quenching of the QDs, minimal adsorption was observed suggesting the methodology has good potential. In addition, we describe the development of a one-pot method for both the synthesis and capping of silicon QDs. Our approach also allows versatile post-synthetic modification of the silicon QD capping to produce a variety of functional groups. Silicon QDs are of interest in biosensing due to their biocompatibility and much lower toxicity compared to II-VI semiconductors.
The results of preliminary investigations toward the design of an optical biosensor instrument for the selective and direct analysis of low copy numbers of target nucleic acids in native form are reported. A concept development prototype was constructed based on a total internal reflection fluorescence (TIRF) configuration and the use of time correlated single photon counting (TCSPC). Selective detection of interfacial hybrid formation was done by identification of luminescence of characteristic (20ns) lifetime from the intercalant fluorophore ethidium bromide associated with nucleic acid hybrids formed at the interaction surface of optical sensor elements. Results of these investigations suggest that detection limits on the order of 107 dye:dsDNA complexes can be achieved when an effective sensor interaction surface of 150 µm diameter is used. The presence of interfacial nucleic acid duplexes at a sensor surface was further verified by thermal denaturation studies. The sensitivity of this concept design prototype was found to be most limited by long lifetime fluorescence intrinsic to the detection optics in conjunction with large amounts of scatter dispersed from the sensor cartridge. Future directions for continued device development are discussed.
Biosensors and biochips can determine the presence of nucleic acid sequences in a test sample through fluorescence detection of hybridization between an immobilized nucleic acid (probe) and a nucleic acid in a test sample (target). The finding that the control of the environment of immobilized single-stranded probe molecules on fused silica surfaces can be used to tune selectivity to facilitate detection of even single base pair mismatches provides opportunities for design of novel biochips. We have typically used silane coupling agents to activate silicon and silicate surfaces for subsequent immobilization of biomolecules for development of optical biosensors. A self-assembled immobilization providing good structural order would be preferred. Studies have been done using thiol-terminated reagents for assembly of oligonucleotides on GaAs substrates. The spacing of Ga and As can be controlled across a surface, and in turn provides a template to control the density of self-assembled oliogonucleotide. Initial experimental work has begun using homogeneous GaAs surfaces, and the homogeneity and surface morphology of immobilized oligonucleotide films grown onto GaAs has been characterized by atomic force microscopy (AFM) and fluorescence methods. Cycles of hybridization and denaturation suggest that the GaAs provides a surface that is stable to loss of immobilized oligonucleotide, but that efforts to protect from non-selective adsorption are essential. Data suggested that the films were of monolayer thickness, and that it was possible to induce the presence of nodules of approximately 10-50 nm in diameter.
We report on development of frequency-doubled diode-pumped ultrashort pulse Yb:KGW laser operating at 520 nm with approximately 200 fs long pulses at a repetition rate of 15 MHz. For ~2 W of absorbed pump power at 980 nm, the laser delivers up to 30 mW of average power at fundamental wavelength of 1040 nm, corresponding to a pulse energy of 2 nJ. The laser radiation was then frequency-doubled in a single pass configuration within a nonlinear BIBO crystal to produce femtosecond green radiation at 520 nm with peak power of ~200 W. The generated second harmonic served as excitation source of optical DNA sensor based on fluorescence lifetime measurements using the time correlated single photon counting (TCSPC) technique.
Immobilization of single-stranded DNA onto fused silica and glass surfaces has been widely used for preparation of fiber optic sensors and DNA microarrays. Fundamental investigations of the orientation, motion and hybridization behavior of immobilized ssDNA is important to understand the design sensing devices. Using computational methods to evaluate molecular dynamics w have simulated a solid SiO2-linker-ssDNA system and studied the conformations assumed by such immobilized material under different physical constraints. We have also evaluated the molecular dynamics of fluorescent intercalating dyes that are attached to the ssDNA by tethers of various lengths, with the goal of preparing a label on ssDNA that will transducer hybridization. Periodic boundary conditions were applied to examine packing properties and nearest neighbor interactions of adjacent ssDNA molecules. Molecular dynamics have been performed at room temperature as well as on heated systems. Solvent effects were taken into account and the modeling assumed the presence of an aqueous environment. The results suggest the various conformations and orientations that immobilized ssDNA can assume, and indicate that the orientation is generally far more that is perpendicular to the interface. Exploration of tether lengths indicates the minimum lengths that are required for a fluorescent label to operate efficiently as an intercalating agent.
Methods for the detection of pathogenic organisms and genetic mutations have evolved to use the selective recognition chemistry offered by nucleic acid hybridization. The immobilization of single-stranded DNA as a 'receptor' onto various solid surfaces that are suitable for optical sensor development is a fundamental issue that determines aspects of selectivity, sensitivity and speed. This manuscript presents an evaluation of various immobilization strategies to attach ssDNA to surfaces, the control of such immobilization to design optical and thermodynamic properties of monolayer films, and suggests approaches for optimization of analytical figures of merit and the potential for quantitative analysis.
In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.
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