Improving the signal sensitivity and photostability of DNA hybridizations on microarrays by using dye-doped core-shell silica nanoparticles

The development of new highly sensitive and selective methods for microarray-based analysis is a great challenge because, for many bioassays, the amount of genetic material available for analysis is extremely limited. Currently, imaging and detection of DNA microarrays are based primarily on the use of organic dyes. To overcome the problems of photobleaching and low signal intensities of organic dyes, we developed a new class of silica core-shell nanoparticles that encapsulated with cyanine dyes and applied the dye-doped nanoparticles as labeling in the DNA microarray-based bioanalysis. The developed nanoparticles have core-shell structure containing 15-nm Au colloidal cores with 95 dye-alkanethiol (dT)20 oligomers chemisorbed on the each Au particle surface and 10-15-nm silica coatings bearing thiol functional groups. To be utilized for microarray detection, the dye-doped nanoparticles were conjugated with DNA signaling probes by using heterobifunctional cross-linker. The prepared nanoparticle conjugates are stable in both aqueous electrolytes and organic solvents. Two-color DNA microarray-based detection was demonstrated in this work by using Cy3- and Cy5-doped nanoparticles in sandwich hybridization. The use of the fluorophore-doped nanoparticles in high-throughput microarray detection reveals higher sensitivity with a detection limit of 1 pM for target DNA in sandwich hybridization and greater photostable signals than the direct use of organic fluorophore as labeling. A wide dynamic range of approximately 4 orders of magnitude was also found when the dye-doped nanoparticles were applied in microarray-based DNA bioanalysis. In addition, the use of these dye-doped nanoparticles as the labeling in hybridization also improved the differentiation of single-nucleotide polymorphisms. This work offers promising prospects for applying dye-doped nanoparticles as labeling for gene profiling based on DNA microarray technology.     

A novel approach for high-quality microarray processing using third-dye array visualization technology

Historically, microarray image processing has been technically challenging in obtaining quality gene expression data. After hybridization of Cy3- and Cy5-labeled samples, images are collected and processed to obtain gene expression ratio measurements for each of the elements on the array. The hybridization process often brings in contaminating noise, which can make correct identification of the signal difficult. In addition, spot intensity levels are highly variable due to the expression differences of different genes, and weak spots are often difficult to detect. These conditions are further complicated by inherent irregularities in spot position, shape, and size commonly found on high-density microarrays, making image processing an often labor-intensive task that is difficult to reliably automate. We previously reported a novel third-dye array visualization (TDAV) technology that allows prehybridization visualization and quality control of printed arrays. Here, we present a new microarray image processing approach utilizing TDAV. By incorporating the third-dye image, we show that overall quality of the microarray data is significantly improved, and automation of processing is feasible and reliable. Furthermore, we demonstrate use of the third-dye image to better quality control microarray image analysis. Both the principle and implementation of the approach are presented in detail, with experimental results.     

Electrical Readout of Protein Microarrays on Regular Glass Slides

A new approach for the electrical readout of microarrays prepared on regular glass slides, using an array of impedimetric transducers (interdigitated electrodes, IDEs) is presented in this work. Impedance detection relies on the use of a urease-labeled immunoassay scheme. Urease is able to produce an increase in conductivity by hydrolysis of the urea substrate, which is measured with the IDEs and directly related to the amount of target analyte. Unlike previous electrical microarrays, the assay does not take place on top of the transducers but on a regular glass slide, which may enable the development of compact multiplexed analytical systems with lower cost per assay. A droplet of solution with the enzymatic substrate is deposited on each transducer of the array, and the microarray is positioned at a short distance (300 μm) so that each droplet wets one transducer and one spot of the microarray. This procedure allows reusing the transducer array for readout of a virtually unlimited number of microarrays. A microarray based on an immunoassay for the detection of a mouse generic protein in a concentration range from 0.03 to 30 μg mL(-1) was carried out to assess the performance of the electrical readout approach. A sigmoid response with a limit of detection of 0.1 μg mL(-1) and a dynamic range of 1 order of magnitude was obtained. A comparative study was also carried out with two well established analytical procedures. First, the urease-based immunoassay was tested in a 96 well microtiter plate using phenol red pH indicator and absorbance detection. Second, the microarray was carried out using the same target protein concentration range but applying a Cy3 label and fluorescence detection. Both assays allowed for the validation of the performance of the presented electrical readout system.     

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry

The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.     

Two-laser, large-field hyperspectral microarray scanner for the analysis of multicolor microarrays

We describe the development and operation of a two-laser, large-field hyperspectral scanner for analysis of multicolor genotyping microarrays. In contrast to confocal microarray scanners, in which wavelength selectivity is obtained by positioning band-pass filters in front of a photomultiplier detector, hyperspectral microarray scanners collect the complete visible emission spectrum from the labeled microarrays. Hyperspectral scanning permits discrimination of multiple spectrally overlapping fluorescent labels with minimal use of optical filters, thus offering important advantages over standard filter-based multicolor microarray scanners. The scanner uses two-sided oblique line illumination of microarrays. Two lasers are used for the excitation of dyes in the visible and near-infrared spectral regions. The hyperspectral scanner was evaluated with commercially available two-color calibration slides and with in-house-printed four-color microarrays containing dyes with spectral properties similar to their commercial genotyping array counterparts.     

Signal enhancement in antibody microarrays using quantum dots nanocrystals: application to potential Alzheimer's disease biomarker screening

The performance of cadmium-selenide/zinc-sulfide (CdSe@ZnS) quantum dots (QDs) and the fluorescent dye Alexa 647 as reporter in an assay designed to detect apolipoprotein E (ApoE) has been compared. The assay is a sandwich immunocomplex microarray that functions via excitation by visible light. ApoE was chosen for its potential as a biomarker for Alzheimer's disease. The two versions of the microarray (QD or Alexa 647) were assessed under the same experimental conditions and then compared to a conventional enzyme-linked immunosorbent assay (ELISA) targeting ApoE. The QDs proved to be highly effective reporters in the microarrays, although their performance strongly varied in function of the excitation wavelength. At 633 nm, the QD microarray gave a limit of detection (LOD) of ～247 pg mL(-1); however, at an excitation wavelength of 532 nm, it provided a LOD of ～62 pg mL(-1), five times more sensitive than that of the Alexa microarray (～307 pg mL(-1)) and seven times more than that of the ELISA (～470 pg mL(-1)). Finally, serial dilutions from a human serum sample were assayed with high sensitivity and acceptable precision and accuracy.     

Aggregation of cyanine dyes at Langmuir-Blodgett monolayers

Cyanine dyes are adsorbed from aqueous solution to Langmuir-Blodgett (LB) films, forming monolayers of Scheibe or J aggregates, whereas the dye in solution is in monomeric state. As well as J aggregates other types of aggregates (H, H^* aggregates, oligomers, dimers) can form and interchange at the LB interface depending on conditions and the structures of the dyes and amphiphiles. Reflection spectroscopy proved to be a useful method for studying aggregates and kinetic processes at the air-water interface as the bulk subphase is not recorded by this method.

Aggregation of dyes seems to be controlled by Coulomb interactions and the surface structure of the interacting monolayers. Cationic cyanine dyes are adsorbed only at negatively charged monolayers (e.g. fatty acids) whereas cyanine dyes with negative net charge form aggregates only at positively charged monolayers (e.g. amphiphilic ammonium salts).

Aggregation of cyanine dyes has also been found at LB monolayers of amphiphilic nucleic acids which have been synthesized for the first time. Aggregates of adsorbed cyanine dyes can be transferred to solid substrates by the LB method and investigated by other spectroscopic methods or microscopy.

Adsorption of cyanine dyes at vesicle bilayers has been reported; this was studied by absorption spectroscopy of vesicular dye solutions. Comparison of these spectra with reflection spectra of corresponding LB films reveals significant differences between the effects in LB monolayers and those in vesicular bilayers.     

Surface ozone measurements at urban coastal site Chennai, in India

The present study was carried out to gain knowledge of current surface ozone concentrations and the effects of meteorological parameters in the highly populated urban area of Chennai, in South India. We have reported measurement results of surface ozone (O(3)) and meteorological parameters from 17th March to 10th October 2005. A photometric ozone analyzer continuously recorded the ozone concentrations at this site. The present study deals with the statistical characteristics of daily and monthly mean ozone levels under different meteorological conditions. The highest ozone concentrations were recorded in ESE-SE sectors. The monthly mean concentrations were higher in May (23+/-14 ppb) and lower in April at this site (10+/-8 ppb). The maximum hourly ozone concentration reached 69 ppb on 21st April.     

Determination of surface-bound-fluorophore orientation by goniometric fluorescence polarization: application to quantification of DNA-chip readouts

We present a polarized goniofluorimeter designed to measure the observation-angle and polarization-dependent intensity emitted by a group of surface-bound fluorescent molecules. We studied two types of surface bonding: In one case, dyes were adsorbed into the surface by spin coating, and in the other, dyes were covalently immobilized to DNA strands. Fluorescent dyes consisted of Cy3 and Alexa546. The substrate was a silicon wafer bearing a silicon dioxide layer. The different samples presented a wide panel of reproducible experimental behavior. By confronting experimental behavior with theory and simulation, we can explain these differences as directly linked to the mean orientation of fluorophores with respect to the surface.     

Preliminary investigation of proton and helium ion radiation effects on fluorescent dyes for use in astrobiology applications

The Specific Molecular Identification of Life Experiment (SMILE) instrument (Sims et al. Planet. Space Science 2005, 53, 781-791) proposes to use specific molecular receptors for the detection of organic biomarkers on future astrobiology missions (e.g., to Mars). Such receptors will be used in assays with fluorescently labeled assay reagents. A key uncertainty of this approach is whether the fluorescent labels used in the system will survive exposure to levels of solar and galactic particle radiation encountered during a flight to Mars. Therefore, two fluorescent dyes (fluorescein and Alexa Fluor 633) have been exposed to low-energy proton and alpha radiation with total fluences comparable or exceeding that expected during an unshielded cruise to Mars. The results of these initial experiments are presented, which show that both dyes retain their fluorescent properties after irradiation. No significant alteration in the absorption and emission wavelengths or the quantum yields of the dyes with either radiation exposure was found. These results suggest other structurally similar fluorophores will likely retain their fluorescent properties after exposure to similar levels of proton and alpha radiation. However, more extensive radiation fluorophore testing is needed before their suitability for astrobiology missions to Mars can be fully confirmed.     

Major aromatic VOC in the ambient air in the proximity of an urban landfill facility

In this study, the environmental behavior of major aromatic VOC (including benzene, toluene, ethylbenzene and xylene, commonly called BTEX) in the ambient air was investigated from a mid-size municipal landfill site located in Dae Gu city, Korea in the winter of 2004. A series of field campaigns were conducted in the course of the study to cover eight different locations within and near this landfill site along with a number of VOC vent systems. The mean concentrations of different VOC species in ambient air fell in a comparable range of at or above a few ppb (e.g., the most abundant toluene 10 ppb). An inspection of the VOC data sets at the studied LF sites also indicated that they are quite analogous to those typically found in other urban areas in terms of their absolute magnitude and relative pattern (e.g., the general dominance of toluene over the other species). In light of the fact that there is active ventilation of landfill gas (LFG: e.g., with their LFG concentrations above a few to a few tens of ppm) in the study area with no other distinct source processes, it can be concluded that the effects of the landfill processes may be as important as other point sources in maintaining VOC concentration levels in certain urban areas.     

Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.     

An optoelectronic nose: "seeing" smells by means of colorimetric sensor arrays

A new approach to general sensors for odors and volatile organic compounds (VOCs) using thin films of chemically responsive dyes as a colorimetric sensor array is described. This optoelectronic "nose," by using an array of multiple dyes whose colors change based on the full range of intermolecular interactions, provides enormous discriminatory power among odorants in a simple device that can be easily digitally imaged. High sensitivities (ppb) have been demonstrated for the detection of biologically important analytes such as amines, carboxylic acids, and thiols. By the proper choice of dyes and substrate, the array can be made essentially nonresponsive to changes in humidity.     

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS)

Surface‐bound microarrays of multiple oligo‐ and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high‐throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning‐probe‐lithography‐based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high‐density arrays with feature densities of 62 500 features per cm². To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA‐ and FLAG‐tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost‐effective microarrays.     

An identification method for altered proteins in tissues utilizing fluorescence derivatization, liquid chromatography, tandem mass spectrometry, and a database-searching algorithm

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiol-specific fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone.     

Fabricating RNA microarrays with RNA-DNA surface ligation chemistry

A novel surface attachment strategy that utilizes RNA-DNA surface ligation chemistry to create renewable RNA microarrays from single-stranded DNA (ssDNA) microarrays on gold surfaces is demonstrated. The enzyme T4 DNA ligase was used to catalyze the formation of a phosphodiester bond between 5'-phosphate-modified ssDNA attached to the surface and the 3'-hydroxyl group of unlabeled RNA molecules from solution in the presence of a complementary template DNA strand. Surface plasmon resonance imaging (SPRI) measurements were performed to characterize the ligation process as well as to verify the bioactivity of the ssRNA microarray in terms of (i) the hybridization adsorption of complementary DNA onto the RNA array to form a surface RNA-DNA heteroduplex and (ii) the hydrolysis of the RNA microarrays with either ribonuclease S or ribonuclease H (RNase H). The hydrolysis of the surface-bound RNA with RNase H required the presence of a surface heteroduplex and, upon completion, regenerated the original 5'-phosphate-terminated ssDNA array elements. These ssDNA array elements could be ligated again to create a new RNA microarray. These RNA microarrays can be used in the study of RNA-protein/RNA/aptamer bioaffinity interactions and for the enzymatically amplified SPRI detection of DNA in the presence of RNase H.     

Detection of Salmonella spp. using microsphere-based, fiber-optic DNA microarrays

Salmonella spp. are one of the most problematic food pathogens in public health, as they are responsible for food poisoning associated with contamination of meat, poultry, and eggs. Thus, rapid and sensitive detection of Salmonella spp. is required to ensure food safety. In this study, a fiber-optic DNA microarray using microsphere-immobilized oligonucleotide probes specific for the Salmonella invA and spvB genes was developed for detection of Salmonella spp. Microarrays were prepared by randomly distributing DNA probe-functionalized microspheres (3.1-microm diameter) into microwells created by etching optical fiber bundles. Hybridization of the probe-functionalized microspheres to target DNA from Salmonella was performed and visualized using Cy3-labeled secondary probes in a sandwich-type assay format. In this study, 10(3)-10(4) cfu/mL of the target organism could be detected after 1-h hybridization without any additional amplification. The DNA microarray showed no cross-reactivity with other common food pathogens, including E. coli and Y. enterocolitica, and could even detect Salmonella spp. from cocktails of bacterial strains with only moderate loss of sensitivity due to nonspecific binding. This work suggests that fiber-optic DNA microarrays can be used for rapid and sensitive detection of Salmonella spp. Since fiber-optic microarrays can be prepared with different probes, this approach could also enable the simultaneous detection of multiple food pathogens.     

Electronic and Molecular Structures of Dyes VI: Estimation of Redox Potentials and Excitation Energies of Cyanine Dyes by Means of HMO Calculation with Regard to Steric Effects.

The correlation has been examined between redox potentials and the electronic energy levels of cyanine dyes, which has been calculated by the HMO method. The agreement between them was quite good for all cyanine dyes studied except monomethine ones. The examination of the discrepancy with monmethine cyanines has led to the following conclusion. In the case of 2,2'-quinocyanine, the stabilization of the lowest vacant electronic energy level and the unstabilization of the highest occupied one have been found to come from the nonplanarity of the dye molecule due to its steric hindrance. In the case of thiacyanine, it has been found that the unstabilization of the lowest vacant level and the stabilization of the highest occupied one arise from the intramolecular interaction between two sulphur atomic orbitals of the dye molecule. The steric effects for quinocyanine mentioned above make the observed reduction potential positive, the oxidation potential negative, and the excitation energy small with respect to the calculated ones: with thiacyanine they make the reduction potential negative, the oxidation one positive, and the excitation energy large. With regard to the above-mentioned steric effects in monomethine cyanine dyes, the HMO method was applied to the estimation of redox potentials and excitation energies of well-known simple cyanine dyes as well as those of unknown cyanine dyes with long polymethine chains.     

DNA detection on plastic: surface activation protocol to convert polycarbonate substrates to biochip platforms

A mild and efficient surface activation protocol to convert polycarbonate (PC) substrates, e.g., plastic bases of compact disks, to biochip platforms for DNA probe immobilization and target detection is described. The preparation procedure (activation, patterning, and coupling) is simple and effective; the on-chip hybridization is sensitive and selective. Particularly, UV/ozone treatment of PC sheets produces a hydrophilic surface with a high density of reactive carboxylic acid groups [(4.8 +/- 0.2) x 10-10 mol/cm2] in less than 10 min at ambient conditions, and no significant aging or physical damage to the substrate is observed. Covalent immobilization of DNA probes via both passive (reagent-less photopatterning and coupling in bulk solution phase) and flow-through (creation of microarrays with microfluidic channel plates) procedures has been demonstrated. Subsequent hybridization shows uniform and strong fluorescent signals for complementary target DNA and allows clear discrimination between fully complementary targets and strands with a single base-pair mismatch. The surface chemistry described herein will facilitate the development of disposable plastic biochips (not limited to DNA microarrays) and the fabrication of biomedical devices that are readable with conventional optical drives.