Comparison of a pump-around, a diffusion-driven, and a shear-driven system for the hybridization of mouse lung and testis total RNA on microarrays

In the present study, we demonstrate the benefits of a shear-driven rotating microchamber system for the enhancement of microarray hybridizations, by comparing the system with two commonly used hybridization techniques: purely diffusion-driven hybridization under coverslip and hybridization using a fully automated hybridization station, in which the sample is pumped in an oscillating manner. Starting from the same amount of DNA for the three different methods, a series of hybridization experiments using mouse lung and testis DNA is presented to demonstrate these benefits. The gain observed using the rotating microchamber is large: both in terms of analysis speed (up to tenfold increase) and in final spot intensity (up to sixfold increase). The gain is due to the combined effect of the hybridization chamber miniaturization (leading to a sample concentration increase if comparing iso-mass conditions) and the transport enhancement originating from the rotational shear-driven flow induced by the rotation of the chamber bottom wall.     

Matrix-assisted laser desorption/ionization mass spectrometry with re-engineered 2,5-dihydroxybenzoic acid derivative

Dihydroxybenzoic acid was modified to three analogues (M2, M4 and M6). The analogues exhibited specific properties that resulted in enhancement of analyte signal intensity with or without addition of iodine compared to the underivatized parent. Addition of iodine to M2, an ester of dihydroxybenzoic acid that had a terminal double bond in the alkyl chain, resulted in peak intensities comparable to the parent, indicating that iodine interaction across the double bond resulted in enhancement although the exact mechanism is not fully understood. No enhancement on addition of iodine was observed for M4, which had a long alkyl chain that contained no double bonds. The alkyl chain allowed micelle formation in solution, which in turn allowed more uniform analyte-to-matrix mixing. The final analogue combined the long alkyl chain of M4 with the double bond of M2 and exhibited either similar peak intensities to that of dihydroxybenzoic acid or better. Micelle formation in solution was examined using spectroscopy and in the solid by reflective microscopy. The standard deviation from spot to spot was considerably lower relative to dihydroxybenzoic acid (RSD 3.4%vs. 14.2%). Unlike dihydroxybenzoic acid, the novel matrix M6 was able to yield characteristic peaks for analytes such as ubiquitin.     

Effect of the Duty Cycle of Flower-like Silver Nanostructures Fabricated with a Lyotropic Liquid Crystal on the SERS Spectrum

Surface-enhanced Raman scattering (SERS) has been widely reported to improve the sensitivity of Raman spectra. Ordinarily, the laser is focused on the sample to measure the Raman spectrum. The size of the focused light spot is comparable with that of micro-nano structures, and the number of micro-nano structures contained in the light spot area (defined as duty cycle) will severely affect the spectrum intensity. In this study, flower-like silver nanostructures were fabricated with a soft lyotropic liquid crystal template in order to investigate the effect of duty cycle. They were observed under a scanning electron microscope, and their spectrum enhancement factor was computed with the obtained Raman spectrum. Then, their duty cycles were measured using a SERS substrate at different locations. A formula was derived to represent the relation between the duty cycle of the nanoflowers and the Raman spectral intensity. This work could promote the actual applications of SERS in high-sensitivity spectrum testing.     

Thermodynamic behavior of short oligonucleotides in microarray hybridizations can be described using Gibbs free energy in a nearest-neighbor model

While designing oligonucleotide-based microarrays, cross-hybridization between surface-bound oligos and non-intended labeled targets is probably the most difficult parameter to predict. Although literature describes rules-of-thumb concerning oligo length, overall similarity, and continuous stretches, the final behavior is difficult to predict. The aim of this study was to investigate the effect of well-defined mismatches on hybridization specificity using CodeLink Activated Slides and to study quantitatively the relation between hybridization intensity and Gibbs free energy (DeltaG), taking the mismatches into account. Our data clearly showed a correlation between the hybridization intensity and DeltaG of the oligos over 3 orders of magnitude for the hybridization intensity, which could be described by the Langmuir model. As DeltaG was calculated according to the nearest-neighbor model, using values related to DNA hybridizations in solution, this study clearly shows that target-probe hybridizations on microarrays with a three-dimensional coating are in quantitative agreement with the corresponding reaction in solution. These results can be interesting for some practical applications. The correlation between intensity and DeltaG can be used in quality control of microarray hybridizations by designing probes and corresponding RNA spikes with a range of DeltaG values. Furthermore, this correlation might be of use to fine-tune oligonucleotide design algorithms in a way to improve the prediction of the influence of mismatching targets on microarray hybridizations.     

Modified Natural Rubber as a Simple Chemical Sensor with Smartphone Detection for Formaldehyde Content in a Seafood Sample

A new biodegradable platform-based sensor for formaldehyde assay is proposed. Natural rubber latex was modified to polylactic acid–chloroacetated natural rubber polymer blend sheets. The polymer blend sheet was grafted using a water-based system with amine monomers as a platform, with a spot exhibiting positive polarity for immobilizing with anionic dye (Acid Red 27). The sensor was exposed to formaldehyde. The color intensity of the dye on the sensor spot would decrease. Using a smartphone with image processing (via ImageJ program), the color intensity change (∆B) could be followed. A linear calibration, ∆B intensity = 0.365 [FA] + 6.988, R² = 0.997, was obtained for 10–150 mM FA with LOD and LOQ at 3 and 10 mM, respectively (linear regression method). The precision was lower than 20% RSD. Application to real seafood samples was demonstrated. The ready-to-use sensor with the proposed method was cost-effective, was portable for on-site analysis, and demonstrated green chemical analysis.     

Distance-dependent emission from dye-labeled oligonucleotides on striped Au/Ag nanowires: effect of secondary structure and hybridization efficiency

When fluorescently tagged oligonucleotides are located near metal surfaces, their emission intensity is impacted by both electromagnetic effects (i.e., quenching and/or enhancement of emission) and the structure of the nucleic acids (e.g., random coil, hairpin, or duplex). We present experiments exploring the effect of label position and secondary structure in oligonucleotide probes as a function of hybridization buffer, which impacts the percentage of double-stranded probes on the surface after exposure to complementary DNA. Nanowires containing identifiable patterns of Au and Ag segments were used as the metal substrates in this work, which enabled us to directly compare different dye positions in a single multiplexed experiment and differences in emission for probes attached to the two metals. The observed metal-dye separation dependence for unstructured surface-bound oligonucleotides is highly sensitive to hybridization efficiency, due to substantial changes in DNA extension from the surface upon hybridization. In contrast, fluorophore labeled oligonucleotides designed to form hairpin secondary structures analogous to solution-phase molecular beacon probes are relatively insensitive to hybridization efficiency, since the folded form is quenched and therefore does not appreciably impact the observed distance-dependence of the response. Differences in fluorescence patterning on Au and Ag were noted as a function of not only chromophore identity but also metal-dye separation. For example, emission intensity for TAMRA-labeled oligonucleotides changed from brighter on Ag for 24-base probes to brighter on Au for 48-base probes. We also observed fluorescence enhancement at the ends of nanowires and at surface defects where heightened electromagnetic fields affect the fluorescence.     

The inter‐ and intra‐operator variability in manual spot segmentation and its effect on spot quantitation in two‐dimensional electrophoresis analysis

Separation of complex mixtures of proteins by 2‐DE is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for identifying alterations in protein expression within a given biological system. Software packages are designed for this purpose. The accurate definition of protein spot boundaries, using a suitable method of image segmentation, is a key requirement for image analysis. It is often necessary for operators to intervene manually to correct mistakes in spot segmentation; therefore operator subjectivity and differences in ability can weaken the analysis. We estimated the error in spot quantification after manual spot segmentation, which was performed by different operators, using two different software packages. Our results clearly show that this operation was associated with significant inter‐ and intra‐variability and an overestimation of subsequent spot intensity, especially when spots were weak. For comparative studies, we suggest separately analysing spots which have been manually segmented by imposing a requirement for at least a threefold difference in spot intensity in addition to use of statistical tests.     

Quantitative measurement of the near-field enhancement of nanostructures by two-photon polymerization

The quantitative determination of the strength of the near-field enhancement in and around nanostructures is essential for optimizing and using these structures for applications. We combine the gaussian intensity distribution of a laser profile and two-photon-polymerization of SU-8 to a suitable tool for the quantitative experimental measurement of the near-field enhancement of a nanostructure. Our results give a feedback to the results obtained by finite-difference time-domain (FDTD) simulations. The structures under investigation are gold nanotriangles on a glass substrate with 85 nm side length and a thickness of 40 nm. We compare the threshold fluence for polymerization for areas of the gaussian intensity profile with and without the near-field enhancement of the nanostructures. The experimentally obtained value of the near-field intensity enhancement is 600 ± 140, independent of the laser power, irradiation time, and spot size. The FDTD simulation shows a pointlike maximum of 2600 at the tip. In a more extended area with an approximate size close to the smallest polymerized structure of 25 nm in diameter, we find a value between 800 and 600. Using our novel approach, we determine the threshold fluence for polymerization of the commercially available photopolymerizable resin SU-8 by a femtosecond laser working at a wavelength of 795 nm and a repetition rate of 82 MHz to be 0.25 J/cm(2) almost independent of the irradiation time and the laser power used. This finding is important for future applications of the method because it enables one to use varying laser systems.     

Electro-chromatography and the movement of complex ions

It is apparent that, in electro-chromatographic separations, the change of area undergone by the initial spot of test solution plays a part in the final interpretation of results almost as important as the actual movement of the centre of the spot.Several interesting facts have emerged from this research concerning the role of the carrier electrolyte on tlie movement, the spot size, and the stability of complex salts.     

Determination of trace tin by solid substrate-room temperature phosphorimetry using sodium dodecyl sulfate as sensitizer

The effects of different surfactants on solid substrate-room temperature phosphorescence (SS-RTP) properties of Sn(4+)-morin systems were investigated. It was found that the SS-RTP intensity of luminescence system was increased greatly in presence of sodium dodecyl sulfate (SDS). A new highly sensitive method for the determination of trace tin has been proposed based on sensitization of SDS on SS-RTP intensity of morin-tin system on the filter paper substrate. The linear dynamic range of this method is 8.0-112 ag per spot (with the volume of 0.4 microl per spot) with a detection limit of 4.0 ag per spot, and the regression equation is DeltaIp=199.7+3.456m(Sn(IV)) (ag per spot), with the correlation coefficient r=0.9998 (n=7). This simple, rapid and reproducible method has been applied to determine the amount of tin in real samples with satisfactory results.     

Analytical investigations on the effects of substrate kinetics on macromolecular transport and hybridization through microfluidic channels

In this paper, a generalized surface-kinetics based model is developed to analytically investigate the influences of the substrate types and the buffer compositions on the macromolecular transport and hybridization in microfluidic channels, under electrokinetic influences. For specific illustration, three typical microchannel substrates, namely silanized glass, polycarbonate and PDMS, are considered, in order to obtain analytical expressions for their zeta potentials as a function of the buffer pH and the substrate compositions. The expressions for the zeta potential are subsequently employed to derive the respective velocity distributions, under the application of electric fields of identical strengths in all cases. It is also taken into consideration that the charged macromolecules introduced into these channels are subjected to electrophoretic influences on account of the applied electric fields. Closed form expressions are derived to predict the transport behaviour of the macromolecules and their subsequent hybridization characteristics. From the analysis presented, it is shown that the modification of the channel surface with silane-treatment becomes useful for enhancing the macromolecular transport and surface hybridization, only if the buffer pH permits a large surface charge density. The analytical solutions are also compared with full-scale numerical solutions of the coupled problem of fluid dynamic and macromolecular transport in presence of the pertinent surface reactions, in order to justify the effectiveness of closed-form expressions derived in this study.     

CaCl2作为异硫氰酸曙红固体基质室温磷光增强剂及其在免疫分析中的应用

有关荧光素的溴和碘取代物的低温磷光性质与取代度关系的研究表明，随着取代度的增大，磷光量子产率与荧光量子产率之比呈现先增大继而减小的趋势，即溴和碘的充分取代物的磷光反而减弱，故用eosin-ITC作标记物的报道很少．化学结构和环境等因素对磷光发射有重要影响；某些已被重原子(Br或／和I)高度取代的试剂，     

Microfluidic chip of fast DNA hybridization using denaturing and motion of nucleic acids

This study presents the effect of fluidic temperatures and velocities on improving DNA hybridization. The efficiency of hybridization could be improved by introducing elevated temperature in the hot region and velocity in the cold region. Compared with the conventional methods, this hybridization microchip was able to increase the hybridization signal 4.6-fold within 30 min. The 1.4-kb single-stranded target DNA was tested. The increasing tendency of the fluorescence intensity was apparent when the temperature was higher than 82 degrees C, and the fluorescence intensity reached an asymptotic value at T>90 degrees C. A mathematical model was proposed to relate the fluorescence intensity of DNA hybridization with the hot-region temperature and the cold-region velocity. Based on these results, the new hybridization chip with the processes of temperature and velocity differences will improve efficiency of DNA detection. The microchip combined with hot-region temperature and cold-region bulk flow velocity effects could provide additional efficiency in DNA hybridization.     

DNA reorientation on Au nanoparticles: label-free detection of hybridization by surface enhanced Raman spectroscopy

DNA sequences attached to Au nanoparticles via thiol linkers stand up from the surface, giving preferential enhancement of the adenine ring breathing SERS band. Non-specific binding via the nucleobases reorients the DNA, reducing this effect. This change in intensity on reorientation was utilised for label-free detection of hybridization of a molecular beacon.     

Color reactions of pyridines with pentacyanoamminoferrate

Trisodium pentacyanoamminoferrate is proposed as a spot test, chromatographic spray or spectrophotometric reagent for pyridines. A 1:1 colored complex of the reagent and pyridine derivative is formed in aqueous solution. The intensity and stability of the color varies with the pH.     

Time-dependence of the polarized intensity ratio in CIDNP investigations

The time-dependence of the polarized intensity ratio within a product molecule has been studied by a kinetic method. The kinetic analysis of polarized product formation shows that considerable changes in CIDNP ratios occur during the reaction but characteristic curves can be obtained under different experimental conditions. A method is proposed to evaluate the relative enhancement coefficients on the basis of these characteristic curves, and to obtain information on the kinetics of the reaction.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

Studies of the Interaction of Platinum Drugs with DNA Using Oligonucleotide Microarrays

Summary: A novel method for the study of the interaction of the platinum drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) with 50-mer oligonucleotides that were printed in high throughput microarray format is introduced. Our aim has been to identify sequence level differences in the interaction of various drug candidates that may serve to enable rational targeting of drugs to specific genes. A microarray of 26 control genes commonly used in oligonucleotide, Affymetrix and c-DNA microarray platforms were microcontact spotted as amine-terminated 50-mer oligonucleotides onto glycidoxypropyltimethoxy silane (GPMS)-modified glass slides. The generalized study format involved hybridization of probes with 10 fluorescently labeled complements as target followed by confocal imaging to reveal original spot intensities. Microarrays were then incubated at 37 °C with hydrolysed cisplatin while in hybridization cassettes, washed in buffer and then scanned again to reveal secondary intensities. We have investigated the influence of cisplatin to stabilize the relative fluorescence intensity via intrastrand crosslinking by studying the impact of varying drug:probe-DNA mole ratio (0:1 (blank), 1:1, 25:1 and 50:1) and annealing temperatures (36, 46, or 56 °C) on retained intensity. ANOVA revealed that 4 of the 10 genes demonstrated (p < 0.0001) the expected result of increased signal retention with decreased temperature and increased drug concentration.     

Hybrid gold/silica/nanocrystal-quantum-dot superstructures: synthesis and analysis of semiconductor-metal interactions

We report on the synthesis and spectroscopic characterization of well-defined hybrid structures that consist of a gold core overcoated with a silica shell, followed by a dense monolayer of CdSe nanocrystal quantum dots (QDs). The dielectric silica spacer of a controlled thickness provides a simple means for tuning interactions between the QD emitters and the metal core. To illustrate this tunability, we demonstrate switching between QD emission quenching and enhancement by varying the silica shell thickness. Synthetic procedures developed here employ a final step of self-assembly of QDs onto the silica shell performed via simple titration of the QD solution with prefabricated core/shell Au/SiO2 particles. This approach allows us to perform an accurate quantitative analysis of the effect of the metal on the QD emission intensity. One important result of this analysis is that nonuniformity of nonradiative rates across the QD ensemble has a significant effect on both the magnitude and the shell-thickness dependence of the emission enhancement/quenching factors.     

Visual scanometric detection of DNA through silver enhancement regulated by gold-nanoparticle aggregation with a molecular beacon as the trigger

A convenient and label-free scanometric approach for DNA assay was designed by integrating a metal-ion-mediated conformational molecular beacon (MB) and silver-signal amplification regulated by gold-nanoparticle (AuNP) aggregation. The strategy was based on displacing the interaction between the target DNA sequence and a competitor Hg(2+) ion with a link DNA sequence. In the absence of the target DNA sequence, a link DNA sequence interacted with the Hg(2+) ions, thus forming an inactive cyclic conformation of the MB. This result led to the poor aggregation of polyadenosine-functionalized AuNPs (A-AuNP). In the presence of a target DNA sequence with a stronger affinity than that of the competitor, hybridization between the link DNA and target DNA sequences turned on the trigger. The polythymidine end of the resulting linear duplex structure could react with A-AuNP, thus leading to a cross-linking aggregation. This aggregation weakened AuNP-catalyzed silver enhancement on a spot substrate. Further, by using scanometric detection, the concentration of the target DNA sequence could be conveniently read out within a linear range from 1.0 to 30 nM. Interestingly, in the same amount of Hg(2+) ions, one-base mismatched DNA showed only 22% of the relative gray-scale intensity for the target DNA sequence at the same concentration, thus indicating good specificity. The designed approach, with the help of the ion-mediated conformational MB, was simple, cost effective, adaptable, and convenient and provided significant potential applications in clinical analysis.