Development of a DNA sensor using a molecular logic gate

This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in the presence of deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as a DNA sensor has been demonstrated.     

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes

OBJECTIVE: To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. STUDY DESIGN: Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. RESULTS: Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. CONCLUSION: Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.     

Sequence-dependent fluorescence of cyanine dyes on microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.     

Synthesis of thiol-reactive, long-wavelength fluorescent phenoxazine derivatives for biosensor applications

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (K(d)) of 435 microM. The fluorescence change for IANR was only 20% and the K(d) was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.     

Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.     

Fluorescent dyes for cell viability: an application on prefixed conditions

In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.     

The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules.

BACKGROUND: The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye-dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy. METHODS: We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO). RESULTS: In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species. CONCLUSIONS: The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission.     

Effective permeability of hydrophilic substances through walls of lymph vessels: roles of endothelial barrier

The wall effective permeability of hydrophilic substances labeled with fluorescent dyes was evaluated in an isolated cannulated rat single lymph vessel through a videomicroscope system. Sodium fluorescein (NaFl; 332 mol wt) and FITC-dextrans (4,400, 12,000, and 71,200 mol wt) were administered into the intraluminal space of the lymph vessels and then excited by a Xenon lamp. Changes in the fluorescence intensity of the dyes were continuously measured by a silicon-intensified target camera through appropriate filters. The net flux of each dye in the wall of the lymph vessels was calculated by the relationship between the fluorescence intensity and the concentration of the dyes. NaFl and FITC-dextran 4,400 in the intraluminal space of isolated rat lymph vessels significantly penetrated the wall of the lymph vessels. FITC-dextran 12,000 in the intraluminal space of isolated rat lymph vessels slightly passed through the lymphatic wall, whereas FITC-dextran 71,200 did not penetrate the wall. Intraluminal pressures ranging from 4 to 8 cm H(2)O did not significantly affect the net flux of dyes used in the present study. After administration of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate into the lymph vessels, the net flux of FITC-dextran 4,400 and 12,000 but not 71,200 was augmented significantly. These results suggest that small molecular hydrophilic substances (< or =4,400) are permeable from the intraluminal to extraluminal space of isolated lymph vessels and that the endothelial cell surface structure may play a barrier role in the effective permeability of large molecular hydrophilic substances (4,400 to 12,000) through the wall of the lymph vessels.     

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.     

Quantitative analysis of fluorescence profiles of chromosomes : Influence of DNA base composition on banding

Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.     

Probing the Orientational Distribution of Dyes in Membranes through Multiphoton Microscopy

Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt μ and distribution breadth γ, are more informative than an average tilt angle 〈φ〉. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.     

On the complex formation of acridine dyes with DNA. VII. Dependence of the binding on the dye structure

The binding constants for the complex formation of more than twenty ring nitrogen-and amino-substituted acridine derivatives with calf thymas DNA were measured by a fluorescence method. DNA quenches the fluorescence of the aminoacridine dyes so long as both amino hydrogens are not substituted. These dyes show an enhancement of their fluorescence intensity in the presence of DNA. Typical representatives of both are proflavine and acridine orange derivatives, respectively. A discussion of steric and electronic influences of various substituents attached to the ring nitrogen and amino groups on the binding led to the concept of different conformations for intercalated acridines without amino groups and the aminoacridines. The electrostatic binding site of the former seems to be the positively charged ring nitrogen, while the binding sites in the aminoacridines are so located that the amino groups are directed towards the negatively charged DNA phosphates.     

Low background signal platform for the detection of ATP: when a molecular aptamer beacon meets graphene oxide

A novel molecular aptamer beacon (MAB) was designed by integrating a single-labeled hairpin-shaped aptamer and graphene oxide (GO). The hairpin-shaped aptamer was constructed with anti-ATP aptamer and another five nucleotides added to the 5'-end of the aptamer which are complementary to nucleotides at the 3'-end of the aptamer to form a hairpin-shaped probe. This newly designed MAB which acts as a low background signal platform was used for the ATP detection based on long-range resonance energy transfer (LrRET). In the absence of ATP, the adsorption of the dye-labeled hairpin-shaped aptamer on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Therefore, the fluorescence of the designed MAB is completely quenched by GO, and the system shows very low background. Conversely, and very importantly, upon the adding of ATP, the quenched fluorescence is recovered significantly, and ATP can be detected in a wide range of 5-2500μM with a detection limit of 2μM and good selectivity. Moreover, when the GO-based MAB was used in cellular ATP assays, preeminent fluorescence signals were obtained, thus the platform of GO-based MAB could be used to detect ATP in real-world samples.     

Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity

By use of a flow cytometer, the fluorescence of cells stained with hematoporphyrin derivative and the fluorescence of plastic beads stained with different dyes were analysed as a function of the intensity of the exciting laser light. The ratios of the fluorescence values of stained and unstained cells as well as of stained cells and beads were sensitively dependent on excitation intensities. As a consequence of this finding, the normalization of cellular fluorescence by use of reference particles needs to be made on a well-defined and reproduced intensity of the exciting laser light.     

Optical spectroscopy of inorganic-organic host-guest nanocrystals organized as oriented monolayers

Oriented and densely packed zeolite L monolayers were prepared on a glass support. The one-dimensional channels of zeolite L, being all oriented perpendicular to the glass and parallel to each other, were sequentially filled by ion exchange with two strongly fluorescent dye molecules. First N-methylacridine (MeAcr⁺) was inserted followed by 3,3′-diethylthiacarbocyanine (DTC⁺). The shorter MeAcr⁺ is oriented perpendicular to the channel axis while the longer DTC⁺ is parallel, due to the constraints imposed by the geometry of the zeolite L channels, as deduced from fluorescence anisotropy of single MeAcr⁺-zeolite L and DTC⁺-zeolite L crystals. The dye molecules can enter the channels only from the top side of the monolayer, since the entrances on the bottom are blocked by the glass support. The resulting ordering has been observed by fluorescence microscopy of single DTC⁺, MeAcr⁺-zeolite L crystals. Conditions were found to suppress the pronounced Rayleigh scattering of zeolite monolayers. Thus high quality absorption spectra of DTC⁺, MeAcr⁺-zeolite L monolayers on glass could be measured at different angles between the incident light and the layer. The results deliver a direct proof that microscopic ordering of the dyes in the channels of zeolite L as well as macroscopic organization of the dye-zeolite L monolayer on the glass support was achieved. Thus a high level of organization was obtained by controlled assembly of the zeolite L crystals into oriented structures followed by subsequent insertion of strongly luminescent dyes.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

Functional binary micropattern of hyperbranched poly(ether amine) (hPEA-AN) network and poly(ether amine) (PEA) brush for recognition of guest molecules

A binary micropattern of anthracene-contained hyperbranched poly(ether amine) (hPEA-AN) network and poly(ether amine) (PEA) brush on gold surface was developed and explored. First, a micropatterned hPEA-AN network array on gold surface was fabricated by photolithography via photodimerization of anthracene moieties, and a PEA brush was subsequently immobilized on the remaining free gold surface areas by chemical adsorption of thiol groups. The patterned hPEA-AN network exhibits selectivity with respect to the adsorption of hydrophilic dyes: Methyl orange is strongly adsorbed, but rhodamine 6G is not, as indicated by the fluorescence response. The PEA brush domain exhibits excellent protein adsorption repellency, whereas the hPEA-AN network layer readily adsorbs protein. These characteristics make the binary hPEA-AN network and PEA brush array sensitive to different kinds of dyes and proteins, which open up pathways to potential applications as microsensors, biochips, and bioassays.     

Effect of TiO2 nanoparticles on some photophysical characteristics of ketocyanine dyes

The effect of titanium dioxide (TiO₂) nanoparticles (NPs) on photophysical characteristics of 2,5‐di[(E)‐1‐(4‐dimethylaminophenyl) methylidine]‐1‐cyclopentanone (2,5‐DMAPMC) and 2,5‐di[(E)‐1‐(4‐diethylaminophenyl)methylidine]‐1‐cyclopentanone (2,5‐DEAPMC) ketocyanine dyes has been studied using absorption, steady‐state and time‐resolved fluorescence spectroscopy. The magnitudes of association constants determined based on modified absorption spectrum of dyes due to the presence of TiO₂ NPs indicate the interaction of TiO₂ NPs with dye molecules. The quenching of fluorescence intensity of dyes by TiO₂ NPs is observed and it follows linear Stern‐Volmer (S‐V) equation. The magnitude of quenching rate parameter suggests the involvement of static quenching mechanism. The involvement of electron transfer process in reducing fluorescence intensity of dyes has been discussed. Also, varying influence of TiO₂ NPs on two dyes is explained based on the presence of different alkyl substituent in two dyes.     

Formation and properties of Langmuir and Gibbs monolayers: a comparative study using hydrogenated and partially fluorinated amphiphilic derivatives of mannitol

The synthesis and surface behavior of a series of nine new hydrogenated nonionic surfactants and their fluorinated analogs, derived from D-mannitol are described. Adsorption monolayers (Gibbs monolayers) were studied by surface pressure (H) measurements as a function of time. For the spread monolayers (Langmuir monolayers), the measurements of surface pressure versus molecular area (A) were performed. For the most hydrophobic amphiphiles at low concentrations, the adsorption at the air/water interface from the bulk solution required extremely long times to attain equilibrium. The A values for two compounds which could be studied in both adsorbed and spread monolayers provided data allowing a direct comparison of the properties of the two types of films formed at the air/water interface. In spite of different mechanisms of formation of Langmuir and Gibbs monolayers, their characteristic parameters were identical, proving the equivalence of these two types of structures.     

Efficient one-pot synthesis of molecularly imprinted silica nanospheres embedded carbon dots for fluorescent dopamine optosensing

A new type of eco-friendly molecularly imprinted polymer (MIP) was synthesized through an efficient one-pot room-temperature sol-gel polymerization and applied as a molecular recognition element to construct dopamine (DA) fluorescence (FL) optosensor. Highly luminescent carbon dots (CDs) were firstly synthesized via a one-step reaction in organosilane, and their surface were anchored with MIP matrix (CDs@MIP). The resulting composite of a synergetic combination of CDs with MIP showed high photostability and template selectivity. Moreover, the composite allowed a highly sensitive determination of DA via FL intensity decreasing when removal of the original templates. The new MIP-based DA sensing protocol was applied to detect DA concentration in aqueous solution, the relative FL intensity of CDs@MIP decreased linearly with the increasing DA in the concentration range of 25-500nM with a detection limit (3σ) of 1.7nM. Furthermore, the proposed method was successfully intended for the determination of trace DA in human urine samples without the interference of other molecules and ions.