Luminescence and stability of aqueous thioalkyl acid capped CdSe/ZnS quantum dots correlated to ligand ionization.

The spectroscopic properties of CdSe/ZnS quantum dots (QDs) were observed to change as a function of thioalkyl acid ligand. Experiments were performed using 2, 3, 6, and 11-carbon linear thioalkyl acids, as well as mercaptosuccinic acid (MSA) and dihydrolipoic acid (DHLA). Bathochromic shifts of up to 14 nm in the emission spectra of QDs capped with these ligands were observed. Similarly, hypsochromic or bathochromic shifts up to 7 nm were observed for a specific ligand in acidic or basic solution, respectively. These shifts could be correlated to the number of ionized ligands and the ability of the ligands to act as hole acceptors. It was also found that differences in quantum yield between the ligands were primarily due to variations in radiative decay rate and not nonradiative decay rate. This indicated that different degrees of QD surface passivation were not responsible for the differences, and that the radiative system must be considered as the sum of the ligands and the QD nanocrystal. The stability of QDs capped with mercaptoacetic acid, MSA, and DHLA towards aggregation at low pH was found to correlate with the pK(a) of the ligands. Spectral shifts were also observed during aggregation. Overall, the luminescence of thioalkyl acid capped QDs appears to be a complex function of dielectric constant, electrostatic or hole-acceptor interactions with ionized ligands, and, to a lesser extent, passivation.     

Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.     

Adsorption and hybridization of oligonucleotides on mercaptoacetic acid-capped CdSe/ZnS quantum dots and quantum dot-oligonucleotide conjugates

Interest in the unique optical properties of quantum dots (QDs) has resulted in the development QD-bioconjugates for imaging and diagnostics. Although these applications are numerous, considerably less is known about the interactions between QDs and biomolecules. In this work, we describe hydrogen-bonding interactions between oligonucleotides and CdSe/ZnS quantum dots capped with mercaptoacetic acid ligands. The strength of the interactions can be modulated by changes in the pH and ionic strength, the addition of formamide, and differences between ssDNA and dsDNA. Fluorescence resonance energy transfer experiments have shown that conjugated oligonucleotides adopt a conformation that lies across the surface of the QD. The hydrogen-bonding interactions also affect the kinetics of hybridization with QD-DNA conjugates and the thermal stability of QD-conjugated dsDNA. The former is analogous to conventional solid-phase hybridization, where stronger oligonucleotide adsorption leads to faster kinetics. With respect to the latter, interactions with the QD surface can sharpen the melt transition and alter the melt temperature of dsDNA. These effects are largely absent when adsorptive interactions are minimized.     

3-Mercaptopropionic acid modified ZnSe quantum dots as the matrix for direct surface-assisted laser desorption/ionization mass spectrometric analysis of peptides/proteins from sodium salt solution

This study describes a strategy of using zinc selenium quantum dots (ZnSe QDs) modified with 3-mercaptopropionic acid (3-MPA) as the matrix for direct analysis of peptides and proteins from sodium salt solution in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The enhancement of detection sensitivity for these biomolecules was due to the adsorption of positively charged peptides or proteins onto the surfaces of negatively charged ZnSe-3MPA QDs via electrostatic interactions resulting in an increase in ionization efficiency for sodium adduct ions ([M+Na](+)). The applicability of the current approach was demonstrated for a variety of peptides, including leucine-enkephalin, methione-enkephalin, HW6, substance P and angiotensin II, and proteins (cytochrome c, myoglobin and lysozyme). Signal intensities of these peptides or proteins can be enhanced by 25-95 times compared with those obtained by LDI-MS in the absence of ZnSe-3MPA QDs. Applying ZnSe-3MPA QDs to serve as the matrix in SALDI-MS is a simple and effective approach for direct analysis of peptide and protein molecules from sodium salt solution without any pretreatment as the peptides and proteins can be successfully detected as sodium adduct ions ([M+Na](+)).     

Chemically induced hairpin formation in DNA monolayers

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.     

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors

The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD₆₀₅) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.     

Biotin-enhanced fragmentation for direct deoxyribonucleic acid sequencing using matrix-assisted laser desorption/ionization mass spectrometry

Fragmentation of synthetic oligonucleotides under the influence of biotin was investigated using 3-hydroxypicolinic acid (3-HPA) as a matrix-assisted laser desorption/ionization (MALDI) matrix. Addition of biotin into the sample enhanced fragmentation of the oligonucleotide between bases. However, when the biotin was tagged to the 5'-terminus of the oligonucleotide, enhancements were observed not only in desorption/ionization efficiency but also in the fragmentation of molecular ions. The protonation/deprotonation process occurs on the tagged biotin is a possible reason for the enhancement in desorption/ionization. Site-specific backbone cleavage fragmentation patterns were observed. The sequences of oligonucleotides can be obtained from their fragment ions. The direct sequencing of a 5'-biotin-tagged 25-mer is demonstrated.     

SOS: A simple interactive program for ab initio oligonucleotide sequencing by mass spectrometry

Mass spectra of oligonucleotides derived from collision-induced dissociation following electrospray ionization provide an effective means of sequence determination, at the 20-mer level and below. An interactive, stand-alone computer program, Simple Oligonucleotide Sequencer (SOS) has been developed for rapid oligonucleotide sequencing from mass spectra, under user control on a residue by residue basis. Modifications can be defined in any combination for the base, sugar or backbone. Sequence ladders can be independently constructed in both the 5' --> 3' directions and 3' --> 5' directions, and graphically compared for homology and overlap. A particular advantage of this method is the ability to easily erase and rebuild alternate subsequences. The program can be used for ab initio sequencing of modified or unmodified oligonucleotides, for rapid verification of sequence, and in studies of fragmentation processes of model oligonucleotide derivatives.     

Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides

This study demonstrates the feasibility of using sol-gel/crown ether hybrid materials as sample substrates that reduce the intensity of the signals of sodium ion adducts of oligonucleotides during matrix-assisted laser desorption/ionization (MALDI) analysis. 2-Hydroxymethyl[15]crown-5 and 2-hydroxymethyl[18]crown-6 were added as dopants during the sol-gel process to generate desalting substrates for MALDI sample deposition. The results demonstrate that the sol-gel/crown ether hybrid materials effectively suppress the formation of sodiated oligonucleotides during MALDI analysis. The largest detectable molecular size for an oligonucleotide was a 100-mer, and the detection limit for an oligonucleotide 36-mer was ca. 20 fmol.     

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometer in electrospray ionization mass spectrometry. 2. Suitability for de novo sequencing

The sequence coverage by fragment ions resulting from collision-induced dissociation in a triple stage quadrupole (TSQ) and a quadrupole ion trap (QIT) mass spectrometer of 10-20-mer oligonucleotides was investigated. While (a-B) and w ion series were the most abundant on both instruments, additional ion series of sequence relevance were preferably formed in the TSQ. Thus, a total number of 83 fragment ions were used to deduce the complete sequence of a 10-mer oligonucleotide of mixed sequence from a tandem mass spectrum recorded on the TSQ. The complete sequence was also encoded in the 28 fragments that were obtained from the QIT under comparable fragmentation conditions. Spectrum complexity increased considerably at the cost of signal-to-noise ratio upon fragmentation of a 20-mer oligonucleotide in the TSQ, whereas spectrum interpretation with longer oligonucleotides was significantly more straightforward in spectra recorded on the QIT. The extent of fragmentation had to be optimized by appropriate setting of collision energy and choice of precursor ion charge state in order to obtain full sequence coverage by fragments for de novo sequencing. Moreover, full sequence information was also dependent on base sequence because of the low tendency of backbone cleavage at thymidines. Tandem mass spectrometry on the QIT yielded redundant information that was successfully utilized to deduce the complete sequence of 20-mer oligonucleotides with high confidence.     

Separation and purification of oligonucleotides using a new bonded-phase packing material

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n-1)-mer present. Baseline resolution of the desired oligomer from (n +/- 1)-mer was achieved under preparative loading conditions, where up to 200-300 micrograms of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.     

An experimental and theoretical study of the gas-phase decomposition of monoprotonated peptide nucleic acids

Peptide nucleic acids (PNAs) are DNA/RNA mimics which have recently generated considerable interest due to their potential use as antisense and antigene therapeutics and as diagnostic and molecular biology tools. These synthetic biomolecules were designed with improved properties over corresponding oligonucleotides such as greater binding affinity to complementary nucleic acids, enhanced cellular uptake, and greater stability in biological systems. Because of the stability and unique structure of PNAs, traditional sequence confirmation methods are not effective. Alternatively, electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry shows great potential as a tool for the characterization and structural elucidation of these oligonucleotide analogs. Extensive gas-phase fragmentation studies of a mixed nucleobase 4-mer (AACT) and a mixed nucleobase 4-mer with an acetylated N-terminus (N-acetylated AACT) have been performed. Gas-phase collision-induced dissociation of PNAs resulted in water loss, cleavage of the methylene carbonyl linker containing a nucleobase, cleavage of the peptide bond, and the loss of nucleobases. These studies show that the fragmentation behavior of PNAs resembles that of both peptides and oligonucleotides. Molecular mechanics (MM+), semiempirical (AM1), and ab initio (STO-3G) calculations were used to investigate the site of protonation and determine potential low energy conformations. Computational methods were also employed to study prospective intramolecular interactions and provide insight into potential fragmentation mechanisms.     

配体对CdTe量子点与BSA的选择性相互作用的影响

以巯基乙酸(TGA)、巯基丙酸(MPA)、巯基甘油(TG)、L-半胱氨酸(L-cys)和谷胱甘肽(GSH)等5种巯基分子为稳定剂, 水相合成了5种CdTe量子点. 以牛血清白蛋白(BSA)作为靶分子, 通过吸收光谱、荧光光谱和时间分辨荧光动力学等手段研究了各种配体分子稳定的CdTe量子点与BSA的直接相互作用. 结果表明, 5种量子点均能有效猝灭BSA的荧光, 其猝灭程度按配体次序为GSH>L-cys>TGA>TG>MPA; 而BSA对不同配体稳定的CdTe量子点的荧光光谱的影响则具有明显的选择性. BSA对TGA-CdTe和MPA-CdTe量子点的荧光先敏化增强而后猝灭下降; L-cys分子由于同时具有氨基和羧基而与BSA的相互作用较强, 因此BSA能显著猝灭L-cys-CdTe量子点的荧光; 而BSA对TG-CdTe量子点的荧光猝灭程度较小; GSH分子的空间效应使GSH-CdTe量子点的荧光被BSA猝灭的程度最小. 吸收光谱和时间分辨荧光动力学研究表明, 5种量子点与BSA之间的相互作用均为静态过程. 探讨了量子点的配体分子结构与蛋白质的相互作用机理.     

Quantitative parameters of cooperative interactions of oligonucleotides within tandem complexes

Quantitative parameters of cooperative interactions of deoxyribooligonucleotides within perfect complementary complexes with a nick in one strand and imperfect complexes containing one mismatched base pair in the nick were obtained. One complementary strand was represented by 22-mer oligonucleotides, while the other, by two short 8-mer oligonucleotides forming a tandem complex with the central part of the 22-mer. In the tandem complexes, the 8-mers form contacts of the following types: 5"-Py*pPy-3", 5"-Pu*pPy-3", and 5"-Pu*pPu-3", where p is phosphate, Py and Pu are pyrimidine and purine nucleosides, respectively, and * stands for a nick. In each incompletely complementary complex, the mismatched base pair in the nick is formed by the 3"-end nucleoside of the 8-mer oligonucleotide and by the nucleoside located in the middle of the 22-mer oligonucleotide. The alkylating 4-[N-(2-chloroethyl)-N-methylamino]phenyl}methylamino group (RCl) is linked through the 5"-end phosphate of the 8-mers (reagents) close to 3"-ends of the 22-mers. The dependences of the limit extents of alkylation of 22-mers (targets) at zero and saturating concentrations of the neighbor oligonucleotides (effectors) on the initial concentration of RCl-derivatives of oligonucleotides (reagents) were used to calculate the association constants K _X of the reagent with the target. The ratio of these constants was used to determine the parameters of contact cooperativity , which characterize the interactions at the junction of two oligonucleotides within the tandem complexes.     

Matrix effect on the analysis of oligonucleotides by using a mass spectrometer with a sonic spray ionization source.

Matrix or impurities remaining in a DNA sample solution after various sample treatment procedures may influence a subsequent DNA analysis. In this work, several matrices were investigated concerning their effects on the analysis of oligonucleotide by using an ion-trap mass spectrometer equipped with a sonic spray ionization source. Inorganic salts of sodium chloride and magnesium chloride depressed the signal intensity by about 50% when the content of the salts was about 10 microM. dNTPs and Taq showed more severe depression on the oligonucleotide. However, Tris, or (hydroxymethyl)aminomethane, intensified the signal intensity, if its content was within an appropriate range. When the content of Tris was about 500 microM, the signal intensity was enhanced by factors of 3 and 5 for the 6-mer and the 20-mer oligonucleotides, respectively. With the existence of Tris, matrix effects from the inorganic salts, dNTPs and Taq were reduced.     

Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry

Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C(18) microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.     

Trace analysis of an oligonucleotide with a specific sequence using PNA-based ion-channel sensors

The gold electrodes modified with self-assembled monolayers of a 13-mer peptide nucleic acid (PNA) probe and 8-amino-1-octanethiol were used for the detection of a complementary oligonucleotide at a femtomolar level using the ion-channel sensor technique. No response to a one-base mismatched oligonucleotide was observed. The electrode surface was positively charged in a pH 7.0 buffer solution due to the protonation of an amine group of the thiol, where the electron transfer between the positively charged marker [Ru(NH3)6]3+ and the surface was hindered because of the charge-charge repulsion between them. Binding of the negatively-charged complementary oligonucleotide to the probe cancels the positive charge at the surface, and provides an excess negative charge at the surface, thereby facilitating the access of the marker to the electrode surface and its redox reaction. Using a 13-mer PNA probe for this sensing mode, we achieved the detection of the oligonucleotide at a femtomolar (approximately 10-15 M) level, improved by five orders of magnitude than the previously used 10-mer PNA probe.     

Application of deuterium–hydrogen exchange to study the secondary structure of oligonucleotide ions in a gas phase

At present, the secondary structure of oligonucleotide ions in a gas phase is almost not understood. One of the main points is the retention of a hairpin secondary structure during ionization. In this work, we used a deuterium–hydrogen exchange reaction in a gas phase at atmospheric pressure for studying the conformational dynamics of oligonucleotide ions formed as a result of electrospray ionization. The exchange reactions of two oligonucleotides, which consisted of the identical sets of nucleotides but differed in their sequence, have been studied. One of these oligonucleotides formed a hairpin secondary structure, but the other did not. It has been found that both of the oligonucleotides demonstrate similar reaction dynamics of deuterium/hydrogen exchange in the gas phase; thereby indicating that the secondary structure has been completely destroyed during ionization.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Effects of oligonucleotide adsorption on the physicochemical characteristics of a nanoparticle-based model delivery system for antisense drugs

Cationic polystyrene nanoparticles, as a model drug carrier system for nucleic acids, are capable of binding negatively charged oligonucleotides by multiple electrostatic interactions. The effect of the adsorption of phosphorothioate oligonucleotides on the physicochemical properties of the carrier system was investigated for uncoated and sterically stabilized latex particles. Turbidity measurements and photon-correlation spectroscopy indicate that the colloidal stability of the nanoparticle-oligonucleotide conjugates is influenced by the number of oligonucleotides adsorbed on the carrier. Especially in the case of the uncoated material, a destabilizing effect has been observed up to oligonucleotide concentrations of 2.7 μmol/g polymer. Strikingly, at higher concentrations the latexes exhibit colloidal stability similar to the oligonucleotide-free samples. These results were correlated to zeta-potential measurements demonstrating a reversal from positive to negative values of the zeta potential with increasing oligonucleotide concentration. The points of zero charge of the particles are in the region of maximum coagulation. These findings were compared to adsorption studies and calculations based on the random sequential adsorption model. It appears that at first the colloidal stability of the carrier systems is diminished with increasing oligonucleotide adsorption, while higher surface coverages lead to a significant reduction in coagulation. At the saturation level the surface coverage can be considered as a monolayer of “side-on” adsorbed molecules and the conjugates exhibit colloidal stability similar to the bare particles without adsorbed molecules. Received: 20 April 1998 Accepted: 16 July 1998