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Minute Papers Due 11/09/2012

Please post this week's minute papers as "comments" to this post. Minute papers should be posted by 5 pm on Friday. Feel free to read your classmate's posts.


Minute Paper #10 (11/08/2012) – Sarah Anciaux

Title: Molecular Beacon Aptamers for Direct and Universal Quantitation of Recombinant Proteins from Cell Lysates

Author: Tan et al.

Journal: Analytical Chemistry

In this paper the authors developed a new method for quantitation of recombinant proteins by using a molecular beacon aptamer that is selective for an anti-His-tag.

The authors developed a new universal detection method by fusing molecular beacons with aptamers. Instead of using a molecular beacon, which usually detects DNA or RNA sequences, the authors took an aptamer that is selective for an anti-His-tag and incorporated FAM, a fluorophore, and a quencher, dabcyl, into the aptamer. When the aptamer is not bound to its target the fluorophore is quenched by the strategically placed quencher, which when in proximity to the fluorophore quenches the fluorescent signal. After the aptamer binds the target, undergoing a conformational change, the quencher is not close enough to the fluorophore to stop emission after excitation, and fluorescence is detectable. By incorporation of a His-tag into a recombinant protein quantitation of the total protein that successfully recombined can be done with ease.

The authors claim to have successfully demonstrated the facile use of this molecular beacon aptamer in recombinant protein detection, but there are some areas of concern. The main concern being that they modified the aptamer that they are using and did not retest the dissociation constant of the aptamer. It is probable that that aptamer is for an anti-His-tag and binds it well, but the modifications done to it make it unlikely that it binds as well in the molecular beacon aptamer state as it did in its native state. A new dissociation curve should be produced to determine if all of the recombinant protein is being bound or if some might be missed. Also, because the developed molecular beacon aptamer is for a tag and not specific to one protein, it seems possible that it could bind to non-specific recombinant proteins if the same tag or something similar was present in another protein or molecule. Further testing of the selectivity and specificity of the molecular beacon aptamer would be beneficial.

Paper: Carbon-dot-based ratiometric fluorescent sensor for detecting hydrogen
sulfide in aqueous media and inside live cells

By: Wu et. al.

Paper: Chem Comm

Link: http://pubs.rsc.org/en/content/articlepdf/2012/cc/c2cc37329g

Earlier in the semester I posted a different paper about the detection of H2S in vivo using the ratiometric fluorescence probe E1. H2S, as I stated earlier, is an important signaling molecule in the gastrointestinal tract. It is an anti-inflammatory aid and is responsible for regulating the contracting forces in the digestive system.

In this paper, the authors introduce a new way to detect H2S in vivo using a different ratiometric fluorescent sensor. Their carbon dot particle, normally fluoresces at 425 nm, but when introduced to H2S, it emits at 526 nm. It is using the ratio of intensities of these two emissions that they are able to quantitatively detect the amount of H2S in a cell. They also quickly tested their probe with several other random molecules in addition to H2S to see if their carbon dot probe was selective for H2S only. They found that it wasn’t affected much by the addition of other molecules. They also were able to show that it does indeed work in living culture cells, so cytotoxicity seems to be ok. The authors did not mention the detection limit of their probe though. I want to know if it is any better than E1 in terms of detection limit. It is essential that this have a lower detection limit, otherwise I see no reason for this to exist. If it isn’t any better than E1 then why use this carbon dot based probe? Also, they were able to show that it worked on living cells, but what about digestive tissue? Will it still work in those cells? Also, the authors didn’t mention anything about competition with other molecules in vivo. I want to see if it can actually quantify H2S levels in actual gastrointestinal tissue cells, not just bovine serum. Finally, again, I don’t see how this would work for actually seeing it in the gastrointestinal tract.

Minute Paper 9
Sarah Gruba
Quantitative Analysis of Neurochemical Panel in Rat Brain and Plasma by Liquid Chromatography-Tandem Mass Spectrometry
Authors: Xiao Zheng, Et al
Journal: Analytical Chemistry
Many neurological disorders involve neurotransmitters such as dopamine, serotonin, and kymurenine. In the past it has been thought that only one or two of these neurotransmitters affects diseases such as depression or Parkinson’s. It has been recently found that there is a dynamic interaction between many neurotransmitters at once which in turn affect each other. The current techniques that were available only focused on a small portion of neuroactive molecules and the authors wanted to create a method to look at more of them at one time. They decided to approach this problem using liquid chromatography electrospray ionization tandem mass spectrometry due to its ability to detect “multiple metabolites from biological samples”.
For their depression model, they chose rats in which were doped with lipopolysaccharide (LPS) through intra peritoneal injection. After 4 or 8 hours the rats were acrificed and their brain cortex was removed. They also received blood from several of the rats at different time points. Using the developed procedure, the authors were able to find significant difference between the control group and the rats injected with LPS. They also found that they were able to monitor the amount of other neurotransmitters accurately and precisely.
There are two experiments that could be really interesting from looking at these results. The first is doing the method in a live animal, and not one that has been killed. The way I see being able to do this, is by taking Bob Kennedy’s method of collecting droplets from the brain continuously that store most of the molecules released (they collect them after injury to see what the cells are releasing) and then going through the author’s mass spectrometry method. This will allow you to actively monitor a live brain and see how the cells are interacting to each other second by second. You also could monitor drug release into the system and how long it takes to reach the brain and the effects of those drugs on the cells of a model with depression or other neurotransmitter diseases. Another interesting experiment would be to do single cell analysis using either TIRFM for non electroactive molecules or carbon fiber microelectrode amperometry to look at how the kinetics of the cell changes with the various diseases in order to see if they have less serotonin and dopamine all together, release less packets of molecules, or take longer to release them. They could also perhaps use the devise Dr. Lane Baker created to look at single cell biological interfaces and incorporate that into sensing the neurotransmitters leaving the cell in vivo.

Title: Promotion of alumina supported cobalt catalysts by iron
By: G. Deo et al.
Journal: The journal of physical chemistry C

In this paper, the group studied the reactivity of alumina supported cobalt, iron monometallic, and Co-Fe bimetallic catalysts. They tested reactivity using carbon dioxide hydrogenation reaction. They said carbon dioxide hydrogenation reaction could be an indicator of the reactivity of important reactions, such as production of long chain hydrocarbon and hydrodesulfurization of crude. For this study, they used X-ray diffraction(XRD), ultraviolet-visible-near infrared(UV-vis-NIR), and diffuse reflectance infrared fourier transform(DRIFT) spectroscopy.

On supported catalysts, Fe, Co, and Fe-Co alloy existed. They confirmed it using XRD and UV-vis-NIR spectroscopy. In addition, they said the method of making bimetallic catalysts also affected the surface structure of catalysts, such as coimpregnation and double impregnation, and sequence of adding Fe and Co in double impregnation. They revealed Co interacts with alumina and forms cobalt aluminate. Impregnation of Fe prevents the formation of cobalt aluminate. From reaction and DRIFT data, Fe first-Co double impregnated catalyst showed the best yield and conversion. Coimpregnated one was the next. Both of them had Fe-Co alloy existed. They revealed that added Fe prevents the interaction between Co and alumina and keeps the reactivity of Co with reactants. Co first impregnated double impregnated catalyst did not have this feature, so there was no improvement in reactivity of CO2 hydrogenation. Also, Fe-Co alloy showed the higher reactivity.

In conclusion, they said Fe-Co alloy helped the carbon dioxide hydrogenation reaction. However, they did not quantify the amount of alloy formed, so they could not get the exact relation between alloy and reactivity. I think using XRD and UV-vis-NIR spectra, they can quantify the amount of alloy. They can make standard bulk alloy and bulk pure metals and compare the intensities and absorbance among them. Then, they can use monometallic catalyst as standard and quantify the alloy and get the normalized reaction rate on alloy. On the other hand, they can conduct this experiment using another reaction that will confirm the enhancement of the whole reactivity of Fe-Co bimetallic catalyst. In addition, they can check the actual weight ratio of metals on alumina, not calculated one, using ICP-MS.

Title: In Situ Precipitation of Amorphous Calcium Phosphate and Ciprofloxacin Crystals during the Formation of Chitosan Hydrogels and Its Application for Drug Delivery Purposes
Authors: Nardecchia, S., et al.
Journal: Langmuir

Nardecchia et al. studied dispersions of amorphous calcium phosphate (ACP) and crystalline ciprofloxacin (CFX) embedded in a chitosan (CHI) scaffold. The authors were interested in developing a biomaterial with the functionality of both ACP, which promotes osteogenesis, and CFX, which fights infections that could develop after surgical implantation of the scaffold. To check if the functionality of the active molecules stayed the same in the scaffold, the authors studied the release kinetics and probed the morphology of the system to see if the components self-assembled into conjugated structures.

The precipitation of ACP and CFX from CHI is induced by increasing the pH of the system to above 6.5. To fully understand the release mechanism, the authors first studied a system of just ACP in solution at different pHs and temperatures by using transmission electron microscopy. At a pH of 6.5 and T = 4 C the precipitate had a dark granular morphology, indicative of ACP. At higher pHs the precipitate formed thin needles that arranged into flower-like structures, which is characteristic of hydroxyapatite (HAp), the crystalline form of calcium phosphate. At all temperatures above 4 C the precipitate was HAp. Due to its higher aqueous solubility, ACP has a better efficacy than HAp.

The authors then studied the morphology of ACP-CFX systems. The resulting precipitate featured a long needle- structure of CFX with the granular ACP particles bound to it. The presence of CFX stabilized the formation of ACP granular particles up to pHs and temperatures of 7.5 and 37 C, respectively. The authors attributed the ACP stabilization to the fact that the crystalline surface of CFX provided a favorable nucleation site for ACP.

The authors then observed the morphology of ACP and CFX embedded in CHI. The scaffolds were prepared by mixing each component in solution, letting it age in an oven to form a gel, and then freezing the gel in liquid nitrogen. In these scaffolds, the CFX and ACP had similar needle and granular shapes, respectively. However, the sizes of these structures were significantly smaller than those found in solution.

Finally, the authors studied the release kinetics of CFX from the scaffold. The data showed that CFX had an initial burst release and was followed by a more sustained release. The authors rationalized that the initial burst was the dissolution of anhydrous CFX crystals, formed during the freezing process, from the surface of the scaffold. Meanwhile, they claim the sustained release was due to the hydrated CFX, which has a lower solubility than the anhydrous, diffusing from the core of the hydrogel.

While the authors present very fascinating results, their studies still leave very many unanswered questions. To corroborate their claim that ACP binds to CFX because of its crystalline surface, they should study the morphology of precipitates of ACP and some other crystalline material, like probucol. Also, the authors don’t discuss the release kinetics of ACP. These studies are crucial because during release the ACP particles could undergo Ostwald ripening and revert to HAp, thereby lowering the efficacy of the scaffold.

Title: Characteristics of low-temperature plasma ionization for ambient mass spectrometry compared to electrospray ionization and atmospheric pressure chemical ionization

Authors: Albert, A.; Engelhard, C.

Journal: Analytical Chemistry

Low temperature plasma (LTP) ionization is a form of ambient desorption/ionization mass spectroscopy and has been used for direct ambient analysis of explosives, drugs and pesticides. However, LTP has not been classified as an ionization technique based on its ionization characteristics and performance compared to more conventional techniques like electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). In this study, the authors compared the relative ionization efficiencies (RIEs) of several compound families obtained from LTP-MS with those from ESI-MS and APCI-MS. The compound families studied in cluded polycyclic aromatic hydrocarbons (PAHs), ionic species, amindes, amines, imides, aldehydes, a nucleoside and a pharmaceutical.

LTP ionization is an attractive option because it is cheap to build and does not require extensive use of gases, power or solvents. It can be used on temperature-sensitive samples like explosives, living cells and other biological materials. LTP-MS instruments can be made as hand-held portable systems.

In order to compare LTP-MS with ESI-MS and APCI-MS, the authors used an internal standard of 4-acetamidothiophenol in acetonitrile, creating a calibration curve from solutions with concentrations varying from 1x10-4 to 1x10-6 molar. The calibration curves for each ionization technique were compared. LTP showed notably higher RSD, but all techniques had similar limits of detection in the 2 micromolar range. All techniques showed a linear dynamic range spanning the range of concentrations studied. Each ionization method also showed comparable analytical performance for detection of the internal standard reference compound.

For nearly all sampled molecular species, all three ionization methods produced the highest intensity signal for either the molecular ion or (M+H)+ ions with little or no fragmentation. This indicated that LTP is a soft ionization source. To calculate RIE, the intensities of protonated dimers were summed with the intensity of monomeric species.

Interestingly, LTP was able to ionize PAH compounds, which ESI is not able to do. Unfortunately, the presence of one or two oxygen atoms in a PAH resulted in an oxidation reaction of the PAH. Other compounds, including pyrene, were detected not only as (M+H)+ but also as a radical. Based on these results, the authors proposed the existence of an additional ionization mechanism that involves single electron transfer.

LTP-MS failed to detect the ionic species, and detected the nucleoside only in the form of its base without the sugar moiety.

Comparison between LTP, ESI and APCI revealed that LTP's ionization characteristics are more similar to those of APCI, especially for amines and aldehydes which ESI cannot ionize to a high extent. LTP showed advantages in ionizing less polar compounds like PAHs and imides. The authors concluded that LTP is limited to small molecular weights but can be applied to less polar compounds.

It is possible that the authors were less successful in ionizing high molecular weight compounds because these compounds are less volatile and harder to desorb. These larger compounds were also more likely to fragment. Further studies could focus upon LTP's ability to ionize less polar molecules.

Marzieh Ramezani, Minute Paper #9

Title: Native Mass Spectrometry Characterization of Intact Nanodisc Lipoprotein Complexes

By: Michael L. Gross and Stephan G. Sligar

Journal: Analytical Chemistry

Nanodiscs are a synthetic membrane system which have been made of lipid bilayers and held by membrane scaffold proteins (MSP). They allow the study of membrane proteins by increasing their solubility. In mass analysis studies, these nanodiscs are usually destroyed before the analysis but in this paper, authors have been used the native electrospray mass spectrometry technique to measure molecular mass and propensity of intact nanodiscs which will provide important information for the next studies.

In this study, the nanodiscs were contained two different scaffold protein: DMPC, and POPC and their native mass spectra recorded by Fourier-transform ion cycloteron resonance (FTICR) mass spectrometer under native conditions. The spectra had two obvious features: broad peaks and nice spaces in between of them. Broad peaks represented nanodiscs and the spaces were probably due to the difference in lipid packing. The charge of each specious was found by dividing the mass of the fragment by the space.

In next step, collisionally activated dissociation (CAD) was employed by gradually increasing the voltage (10 – 190 V) and the spectra were recorded. It was found that nanodiscs structure remains intact when CAD voltage goes up to 70 V. This condition was sufficient enough to dissolve the nanodiscs. Upon increasing the voltage to 100 – 130 V, nanodiscs started to lose the small amount of lipids but the general structure remained undamaged. Finally with 130 V, the nanodiscs structures were fully destroyed and just small lipids mass was found in the spectra.

I think their work just provide preliminary results about the gas phase structure of nanodiscs. They could have done more completed and advanced experiments to determine the exact structure of nanodiscs and elucidate them.

Seminar: Structure and Dynamics of Interfacial Water
By: Mischa Bonn
Date: November 1, 2012

Some of the challenges current spectroscopic techniques must overcome are developing suitable methods for analyzing chemical phenomena operating at the femtosecond time scale. In this seminar, Dr. Mischa Bonn described current techniques utilized to assess structure and dynamics at the water-air interface.

2D sum frequency generation spectroscopy (2D-SFG), by accessing discrete spectral features and fs resolution, provides considerably more information than 1D IR and was therefore utilized to analyze the molecular dynamics along interfacial water. Vibrational sum frequency generation (VSFG), a technique that irradiates a material with two different electromagnetic frequencies (one in the visible and the other in the IR spectrum) and releases one sum of the two frequencies, was used to monitor molecular dynamics and charge transfer along the air-water interface. One of the most intriguing aspects of 2D-SFG interfacial water analysis is the “pumping” frequency is not surface specific, while the probe frequency is. This provides selective analysis of the molecular environment about the water surface, while ignoring those chemical phenomena of bulk water.

Much of Dr. Bonn’s work concerning this project was directed towards understanding vibrational phenomena of water. Initially, he hypothesized that interfacial water behaves somewhat like ice in terms of its vibrational modes. This is counter-intuitive, as ice, even at freezing temperatures, maintains a nano-scale film of liquid phase water. This film is explained by a thermodynamic gradient between the ice-water and water-air interfaces. Dr. Bonn proposed a vibrational coupling hypothesis, which included a water stretching and bending overtone coupling series. In order to test this hypothesis, deuterium isotopic substitution was done and proved to change little of the coupling character of the modes. This suggested surface water structure is not comprised of ice-like structures, because deuteration would negate splitting of coupled vibrational frequencies. Also, his work concerning ultrafast 2D spectroscopy revealed water molecules transfer energy via weak-strong hydrogen bonding reorientations, but not as efficiently as bulk water.

Dr. Bonn wished to understand interfacial water’s rotational character via polarization fs-IR spectroscopy, in which he studied the anisotropy of OH bonds. Anisotropy in bulk water cannot be possible, as the orientation of OH groups is not maintained due to enormous opportunities for hydrogen bonding. In addition, SFG suggested rotationally free surface OH groups are 3-4 times faster than those in bulk water, which is intuitive considering the water-air interface is not spatially hindered as much as bulk water. Lastly, Dr. Bonn explored nuclear quantum effects on hydrogen bonding energies. Using an orientational distribution for H2O, D2O and HDO, oxygen-hydrogen bonds are more likely to orient themselves toward the vapor phase than oxygen-deuterium bonds. This may be explained by stronger hydrogen bonding by OD groups than OH groups.

I found his talk very intriguing, as it attempted to elucidate an enormous amount of information concerning effective characterization techniques of a material we think we know well. I think future work should attempt to characterize ionic solutions via 2D-SFG in order to see how interfacial water’s vibrational behavior changes.

Title: Transition Metal Oxide Work Functions: The Influence of Cation Oxidation State and Oxygen Vacancies
Author: Mark T. Greiner
Journal: Advanced Functional Materials

In this work oxygen vacancies are probed for their effect on the work function of transition metal oxides. The importance of the work function for electrical devices is paramount where charge transfer must occur across material boundaries. The author claims that there is a non-obvious general relationship between cation oxidation state and the oxide’s work function which can be traced independently from other influences. UPS and inverse photoemission spectroscopy (IPES) and XPS among other instruments are used to characterize the band energies and binding energies of the films.

Many factors contribute to transition-metal oxide chemical and electronic properties: multiple stable oxide states, surface dipole (affected by crystallographic orientation, surface termination, adsorbates, etc.), impurities, crystal structure, defects, etc. Greiner et al. sets up experiments to measure the surface dipole using the archetypal d0 transition metal oxide MoO3 by vacuum sublimation of MoO3 powder at 550pC. He states that this experiment allows observation of the materials changing work function without changing crystal structure.

Other oxide films, for determining work function, were first sputtered from pure metal targets onto silicon substrates and subsequently thermally oxidized for 3+ hours around 300oC. Samples were later vacuum annealed and ion bombardment was used to generate oxygen vacancy defects in the films (.5k, .5uA ion sample current -pulsed 5sec) of varying degrees. A model is generated to explain the theory.

Surface dipole results from UPS measurements show that with the increase in oxygen defects the O 2p valence band shifts to a higher energy and a new occupied state appears within MoO3's bandgap. The author proposes that this gap state arises (around 1eV) due to Mo reduction to maintain charge neutrality. He reinforces this position with XPS results showing an increase in reduced Mo 3d spectra.

In the results for the sputtered metal oxide films, 9 plots were shown of different metal oxides with various degrees of oxidation states vs. the associated work function energy (determined via UPS). There is a general trend of lower work functions for pure metal films to higher work functions for increasing metal oxidation states, but there is plenty of scatter in the data. Greiner et al. claims this is because work function depends on oxide thickness for thin films as a result of defects near the metal/metal-oxide interface up until a certain thickness, when it plateaus.

I think the Surface dipole experiments were well controlled to prevent adsorption by other atmospheric molecules. However, it is well known that sputtering and annealing films produces different structured metal oxide quality compared to evaporation. By creating the surface dipole MoO3 films via a different film deposition method than the vacuum annealing method used for oxidizing the other metal films, he is overlooking the effect that the baseline crystal structure has on the overall work function of the material.

Although the experiments for ion sputtering to preferentially remove oxygen from films was done at low energy, there exists the possibility that film structure was modified during this process. It is well known that surface structure changes during sputtering from localized heating, as well as “knock-on” effect and diffusion of surface elements with smaller atomic radii. He considered none of this in his work.

Minute Paper #9 (11/9/12) – Matt Irwin
Title: Polarity-Switching Top Coats Enable Orientation of Sub-10-nm Block Copolymer Domains
Authors: Bates, C., et al.
Journal: Science

The development of block copolymer thin films with vertically oriented domains spaced on the order of sub-10 nm is desirable for a variety of applications such as organic electronics, precision templating and lithography, and nanoporous membranes. In order to achieve these small size scales, lamellae-forming block copolymers must have a very large interaction parameter (χ) between the polymer blocks. However, when these block copolymers are deposited as thin films onto substrates, this large degree of incompatibility between the blocks results in unequal interactions between the blocks and top and bottom interfaces. This unequal interaction leads to one of the blocks preferentially wetting the interface, resulting in horizontally, in plane oriented lamellae domains. This problem is often mitigated on the bottom interface by neutralizing the substrate via a thin layer of a polymer that interacts with each block of the copolymer equivalently. However, neutralizing the top interface is not as easy, as any solvent used to solubilize the top coat material would also solubilize the block copolymer. In this paper, the authors describe an elegant method for developing vertically oriented lamellae in which the top coat material is deposited in in a polar form and then chemically reacts upon baking to form the desired neutral interface. Techniques used include scanning electron microscopy (SEM), thin film infrared spectroscopy (IR), spin coating, and free radical polymerization.

For this study, the authors synthesized two lamellae-forming block copolymers: poly(styrene-b-trimethylsilylstyrene-b-styrene) (PS-PTMSS-PS) and poly(trimethylsilylstyrene-b-D,L-lactide) (PTMSS-PLA). The top coat was a polymer consisted of three co-monomers: (1) maleic anhydride, which is capable of polarity switching; (2) norbornene, a stiff unit which increases the overall glass transition temperature (Tg); and (3) a third co-monomer which allows tuning of the top layer’s interaction energy. The authors dissolved this polymer in ammonia hydroxide and spin coated the solution onto the surface of the block copolymer. Ammonia hydroxide causes the maleic anhydride units to ring-open and to form an ion pair with local ammonium ions. The films were then baked, causing the ammonium to evaporate and the anhydride to ring-close, as evidenced by the disappearance of COO- stretching bands at 1400 and 1560 cm-1 and appearance of C=O bands at 1775 and 1850 cm-1. The samples were annealed for approximately one minute at a temperature greater than the Tg of the block copolymer but less than the Tg of the top coat, and the top coat was subsequently removed with aqueous ammonium hydroxide. The samples were then etched using an O2 reactive ion etch and imaged using SEM. All samples exhibited vertically oriented domains; the PS-PTMSS-PS and PS-PLA exhibited a line spacing of 14 nm and 9 nm, respectively, which compare well to bulk spacings of 15 nm and 7 nm. The authors then describe a model which predicts the film thicknesses for which vertically-oriented lamellae are preferred as a function of top coat interfacial energy, providing criteria for future film design.

This study provides a fascinating new method for developing well-oriented lamellar domains of silicon-containing polymers. I think that it would be interesting to take this project one step further and attempt to develop well-aligned, well-oriented domains. This alignment should be possible by loading the sample into a rheometer and shearing in the direction of preferred orientation direction while annealing. This technique has previously been used to align bulk polymer samples. Domains oriented in this way could serve as templates for advanced materials such as nanowires which require order over long length scales.

Minute Paper #8
Tian Qiu 4651092
Title: Reversible Photoswitching of Spiropyran-Conjugated Semiconducting Polymer Dots
Authors: Yang-Hsiang Chan,† Maria Elena Gallina,† Xuanjun Zhang, I-Che Wu, Yuhui Jin, Wei Sun, and Daniel T. Chiu*

This article demonstrated a novel design and synthesis of photoswitchable polymer-dots (Pdots) by conjugating the photochromic couple spiropyran/merocyanine (SP/MC) onto poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1′-3}-thiadiazole)] (PFBT). Ultraviolet irradiation can change the spiropyran into merocyanine form, which is a quencher in the Foster resonance energy transfer (FRET) process with PFBT. By applying visible light, the MC form returned to SP form, which recovered the PFBT fluorescence. Moreover, they conjugated biomolecules onto the surface of those Pdots and demonstrated their cellular and subcellular labeling, showing the potential of this photoswitchable system in those super-resolution microscopy techniques based on the localization of individual fluorophores, while other photoswitchable system suffered from large particle size, limited brightness, low quantum yield, etc..
To synthesize these Pdots, first, they covalently bound SP onto fluorene moieties of PFBT. Different substances in synthesis were used to explore the optimal method, giving out the result that PFBT functionalized with 50% of carboxyl groups and SP functionalized with hydroxyl groups were the best reactants. The next step was to blend the carboxyl-terminated polystyrene polymer (PS-PEG-COOH) with the matrix of PFBT-SP to increase the carboxyl group on the surface of Pdots to assist biomolecule conjugating. Nanoprecipitation under vigorous sonication was used for Pdot formation. The PFBT-SP Pdots were then conjugated to Streptavidin for cellular labeling.
The efficiency and repeatability of the SP-OH photoswitching cycle and the emission spectra of PBFT-SP-streptavidin Pdots were characterized. The fluorescence of PFBT-SP-streptavidin Pdots at about 540 nm was significantly reduced by the MC molecules and its appearance demonstrated that the quenching of PBFT emission was caused by FRET with MC. Also, a very high absorption efficient was characterized, ensuring the achievement of high contrast level by the low excitation power in super-resolution imaging microscopy techniques. At last, they specifically labeled the surface of live MCF-7 cells. The image showed that the Pdots specifically targeted the surface membrane and a good photoswitchable performance was obtained.
This paper reminded me of a paper using quantum dots for cellular labeling. Apparently, Pdots are better than quantum dots because of their biocompatibility. And Pdots are easier for modification than quantum dots. The design of this photoswitchable system is fascinating. However, this paper didn't talk much about the temporal resolution of this photoswitchable system. It should be a very important factor for its application in the super-resolution imaging techniques such as STORM. More experiments could be perform to explore the temporal response of this photoswitchable system.

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Tu sei grande con le parole. Sono sicuro che hai lavorato davvero duramente per questo articolo, e si vede. Sono d'accordo con un sacco di materiale. Ho apprezzato molto questo e io sarò per tornare più.

Good day! This is my 1st comment here so I just wanted to give a quick shout out and say I really enjoy reading through your blog posts. Can you recommend any other blogs/websites/forums that cover the same subjects? Thanks a ton!

"Weet je echt je spullen ... Keep up the good work! "

Ohh! Mais c'est un très beau message!

oeps, ik lees dit bericht een beetje laat, mijn opdracht net vorige week ingediend en ben ervan overtuigd dat het zou zijn geweest een betere classificatie als ik eerder op deze website gekomen. Wat jammer. Toch bedankt.

Dude .. Ich bin nicht viel in Lesen, aber irgendwie hab ich zu viele Artikel auf Ihrem Blog zu lesen. Es ist erstaunlich, wie interessant es für mich Sie sehr oft besuchen ist. -

Bedankt voor het informatieve functie. Ik ben blij dat dit bericht heeft me geholpen bespaart vele uren surfen op andere gelijkaardige berichten gewoon om te vinden wat ik zocht. Alleen wil ik zeggen: Dank je wel!

LOL! That was really funny!

Hey! Je suis Angela. Inscrite à avoir des discussions franches et en savoir plus sur la recherche d'une carrière dans la technologie. Je vais poster et poser des questions un peu comme je me prépare à ce que le monde a en réserve pour moi.

Ich neige dazu, zu bleiben weg von den folgenden Blogs, in Weltuntergangsstimmung durchdrungen werden und zeigen Menschen, die die Hoffnung aufgegeben haben und nur darauf warten, für die "endgültige" Schuh zu fallen.

@ Alex, ce commentaire n'est probablement pas faite par un lecteur régulier, mais plutôt par un lecteur de passage qui a trébuché sur le poteau par accident.

Heya! Sólo quería preguntar si alguna vez tienes algún problema con los piratas? Mi último blog (wordpress) fue hackeado y terminé perdiendo meses de duro trabajo por ninguna copia de seguridad. ¿Tiene alguna solución para proteger contra hackers?

Io sono un anziano pensionato, e spero di essere di qualche aiuto qui

Hey - nice weblog, schaut sich um einige Blogs, scheint eine ziemlich gute Plattform, die Sie verwenden könnten. Ich bin derzeit mit Wordpress für ein paar meiner Seiten, sondern schauen, um 1 von ihnen mehr als eine Plattform ähnlich wie bei Ihnen als ein Probelauf verändern. Etwas in spezifischen würden Sie darüber empfehlen?

hallo aan u allen, ik ben ook nieuw op de forums. Al heb ik een paar ogenblikken geleden kwam, ben ik op de loer het forum op dit moment, het krijgen om te weten wat er gebeurt.

Merci pour votre apport sur ce sujet.

Salut les gars, blog commentant sur internet est une façon facile de partager commenter les uns les autres. Cette méthode peut fermer les gens dans le monde entier dans la sympathie et l'empathie. Je crois blog commentant est également une source de savoir les opinions des autres

Hola chicos! He estado en al PC, desde que era un niño. Acabo de recibir un nuevo trabajo como técnico de escritorio en una empresa de contabilidad. Sé que voy a tener muchas preguntas para ustedes, pero voy a empezar con un simple hola!

Hola, no estoy seguro de esto. ¿Podría proporcionar alguna información adicional?

Ich habe auch mit Autos ein bisschen spielen, lernen einige Mechaniker arbeiten. id mag in der Lage sein Motormanagement abzustimmen, entweder eine Aktie ecm zwicken oder tune eine vollständige auf Standalone, also wenn jemand das Zeug hier kennt mich PLEAASSEE Punkt, wo man schaut, um loszulegen, weil im so verloren

Analytical Spectroscopy: Minute Papers Due 11/09/2012
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550 の黒い同等 SA デルリン 507 白として選択されています。デルリン 570 位置は 15% または 20% コンテナーおそらく繊維フィラー。これらの web ベースのデザイナーのトートバッグ店、多くのブランドの広大なコレクションを持っているし、一年中、としてすべてのビットに割引価格を提供します。さらに、彼らが本物であると言っているこれと共に各ブランドの署名されていない改ざん。
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ルイ ・ ヴィトン マヒナ バッグは厳密に目標にかかわって弱虫牛革で購入できるそれレーザーとジーンズ素材でテレビ シリーズの衣服シリーズの他の古典的なモノグラム車で構成するこれは意味するそれの高い知識によって設計された、穿孔のエディション黒、ピンク、白、真鍮シルバーとゴールド色の永久的な期間を調べている、LV は一連を持っていた私たち有意義かつシックなマヒナ、その美しさを失うことがなく控えめといい。これは時々 ハリアー VTOL のジェット戦闘機です。だから、VTOL 機が手段、個々 のパイロット、それ職業行う総合的なヘリコプターや飛行機、あなたの決断をされています。つまり、それを脱いですることができます、put の任意のヘリコプターの着陸、それ以外の場合を増やすことができますそれは戦闘機としてどちらか優秀な。

彼らは中に属性を実際の 1、また抵抗力がある費用低利益そっくりパーソナライズしているより多くの人です。多くの場合良いルイ ・ ヴィトン価格心配開発者世界。イヤホン タイプとハンドバッグ ユーザー コレクションを所有しています。彼女の商品は、ちょうど約すべてが断続的に公式ユーザー設定カテゴリ cal 王、しかしまた役割内部感覚として実質的に同一の条件を使用して使用されます。

服ファッションの選択している従ってレベル数十年前、しかしカップル トリー ・ バーチ テニス靴なしを今日開発されるでしょう。このようなストアのと、専門家で時計を見つけます。もちろん、コンサルティング、教育がある使用料を納付する必要があります。まだこのされて価値があるので、たびに、専門家が専門的に行う必要があります。木星、または古代ギリシャ語の言語、ヘラ (ゼウス夫人) と同じです。任意のイベントで、ジュノ Hera は神および女神の女王です。
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* 1980 年代、おそらく秋はおしゃれな滞在を肩に強い傾向によって書かれた根の見直し。強力な肩は本当に完全に実行中の動向、特に冬の服です。肘パッドを伝える非常に豪奢な一見を別の追加ホイール群衆を感じさせてくれます。3. 論文を決定するあなた自身のウェブサイトと組み合わせてビジネスあなたの評判とそれをする必要があることができる認識する出資の目的します。選択技術と上司のラベルと正確に何を colligate オンライン ツールを仮定する傾向があります。

デブラ オンライン ビジネス マルシア コンテンツ 1940 年 10 月の終わりに、Greco イタリア矛盾の中に始めたブログでセレナ Diamandi されていたコニツァにアルバニア語ギリシャ語ボーダーをすでに。このため、この選択の履物仕事の流れ常にトーリー トリーバーチ小売り店最近の靴ブーツの流行を意図して関連する情報の範囲内。彼らは、する必要があるためにのように、最も関連性の高い開発のみの浅黒い肌とブーツ、トリー ・ バーチ ウェブサイト アスレチック シューズ関数の打撃の足の期間。
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大きい船員ズボン、ポップ ズボン、midi の長さのドレスとのサンドレスと一緒にペアを示します。サルヴァトーレ ・ フェラガモ フラットの Menwear に触発されたこの来年このオックスフォード ローファー帰るをする必要があります。さえカントーではビンテージ キャップつま先オックスフォード、任意旅行 moc やよく知られている来年かもしれない履物である従来の赤いセント ローファーを好むがそれは引き続きホット傾向同様に転落。利点をその可能性はすでに: 豊富な同一の写真の量の美しく印刷された並べ替え。小規模なオフィスのカラー漫画を作成するための文字します。このビジネス間の源の環境のアセンブリはポリエチレン、偉大な能力を誇る、米国国務省の標準低下他の多くの材料が良いようなものです。
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そこは通常周辺 A。オンライン委託店メンズその女性のデザイナーの服の上に実現するために得られました。あなたが、画像の近くに滞在する場合そのプラダ コーデュロイやテレビ オンライン中古実際の結論があるし、前年を含むフードを介してエドハーディの女性のトレーナー シックな服装を得ることを格納します。
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休暇要素に関連付けられている部分の分け前はスタンバイと呼び出しの時間プロパティの特定の量を財産の所有者を与えられます。1/13 リスト意味特定の所有者の 1 泊旅行; 家 20 8 日を楽しんでいます1/6 レビュー 8 週間毎年を人を与えられます。いくつかの部分のプロパティ; 夏の期間のための回転の家販売人週間作業スケジュール、セットを持っています。
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卸売メーカーのネックレスの宝石サプライヤーの開発予算よだれかけネックレスを購入、良い割引を入手できます。重要なラムスキン ノックでエルメス財布市場にデザイナーの支出をすすぐし、見つける素晴らしい支払わなかったをこれまで知ることはできません。クラシック ツイード、均一と別のエルメス財布のペアまたはマイル乗算プラットフォーム ペアの靴を見つけた見てトレンド セッターを発掘これまで。エルメス handrao、行動を取るあなたとしてその他のアクセサリーと一緒により多くの選択肢のレプリカで以下を過ごすとき衣装としても。

別の人とハード スタイルの銀食器、のみすることができますを集めているだけでなく魅力関連付けられているティファニーの銀食器を完全に表示を見つけるに一致します。カスタム荷物は販売事業、スポーツ チーム、アイデアや意見から大幅に渡すのも気に入った方法です。これは、袋を伝える写真とメッセージは頻繁に印刷される、または、縫製することができます魅力的な素晴らしい上の作業を開始良いリグを描画します。
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型の財布と自信を持って現在特性および優雅を定義することができます。現在、マルチ レベル マーケティング努力の間にわずかな接続に不満ですか?あなたがドン、最終的にはまだ、長い永遠に置くことがある十分なクライアント。一人で入るずっと。少しの小さなバーと 1 つの専門家が、あなたのランニング シューズ。
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我々 はおそらくなる期待あなたの会社はミハエル Kors のために直面してあなたが任意の競争力のある逆風を軽減する手順を説明します。あなたの責任間の勝者ルイス (コーチのリード デザイナー兼重要な商業) を譲る彼の準備で使用されるコーチ サプライヤー Lew フランクフォートをほめます。
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可能性が高い場所を選ぶ新しいソニー hula-hula トム Kors ウェストン バック パックは、非常に最初に正確な。非常に次の時間、しかし、不十分な Sarge を表示するいくつかの他の人を取得します。私のフィートをホーム取得する修飾移動彼および彼からの機能するのに十分な長さではない!いくつかの練習などを行った外。ÂI の絶対にないアイデアを持っていた何彼が行うような我々 はそれを得た。
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事務所あたり見て他の生産の場所と共に中国住宅技術の方法を監督します。完成品は米国で販売のための惑星の多くのものに運ばれます。DLF エンポリオは 5 つのアイコンのスタイルの任意の時点のジャンルの中から Mohit Gujral 建築家に設計されています。いくつかの天窓、開放性の概念を持っている概念の球 2 つの息をのむアトリウム コートヤードは不可欠のテーマです。
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すべての残りは十分な長さの結果としてのぞき見を一般的なシフォン ハンカチのまたは彼らが偽ることができます。エメリッヒ スティーブン ・ スピルバーグをしばしば羨望が、映画製作者が、恥ずかしいの成功エッジの舐める可能性があります ("我々 より実質的な設定を必要とするつもりだ、キューザック回っていくつかの時間)。無臭のポケット ポーチは 3 の異なる色合い、赤、青緑、緑の自然で購入できます。* 簡単にアクセスできますポンド覚えておく能力を容量としての負荷としての無臭の実用的な安全: 22 ポンド ギャップ。
スニーカー new balance

あなたのための 4 番目の質問は、あなたはセルフ スターター タイプですか?ほとんどの人々 の周りスティックからあなたの時間と努力を個々 の描画を行うと与えられた同僚の支援についてのアドバイスですか?まあそれを信じる場合自分であまりにも自己意識を取得する可能性があります上の可愛い人。Y 男には暗い人工の革のジャケットが含まれています、それは長いこと、ニット ベージュまたはオフホワイトのスカーフ、独自の要件のペア エース予備の一見。騒々しい。ばね 2010 年グッチは助言するその輝かしい歴史と繊細なデザインを再び新しい同じ靴で習慣的な考え一緒に袋可能性があります。
ニューバランス m1400

黒になりますやが付属して靴エキスを通して染め彼らタンで購入することができます。場合は決してイタリアの運動靴のいくつかを所有している、これはすぐに持っている動揺を購入することはできません。エルメス バーキンのブランドに関連する典型的な高品質のシンボルになるし始めているし、人に関して魅力を得ることができますどのように rrn エルメス バーキン バッグに位置を投資することができますを取得します。革製品の男性については特に、焦点を当て賢明であり、その前面より良い製品ラインを作っている彼女のデザインのキャンプと同様。何かのための手数料を求める場合彼らは最も電卓に様々 なパンチし、あなたに機能可能性があります。
トリーバーチ ブーツ

インスタント使用さらにキットの無機綿と良い興味深い女性デザイナー財布に十分な場合は。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。Gridwalls とは私は主にポスターと共にそれの材料の沈没によって簡単な言葉でこの 1 つのストアの外観を変更することができる、どんな機器本当にことができると思うは !弓罫線、選択肢は無限にある私のグリッドを作成するに関するアンドアウトを織り可能性がありますの!それらの gridwall は Gridwall 来るとの選択を要求、するのであなたに入れる約 2"を実際の壁と、グリッドがあります。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。だから良いラップトップ バッグの選択は、優れています。1 つのエネルギー分野好意の基本的に新約聖書を使用してこれらの作品の開発はしばしばデザイナー アフター マーケット クラッチ。Gridwalls とは私は主にポスターと共にそれの材料の沈没によって簡単な言葉でこの 1 つのストアの外観を変更することができる、電球何でもよく思うは !私は織りリボン アンドアウト可能性のボーダーそれらを公開する私のグリッドのすべての使用実際になる無限 !Gridwall Gridwall マウント、あなたの周りで可能だと外側の装備する必要がありますので 2「壁ともグリッド; 処理間で選択するには2「名前の装飾にスペースの配列です !暖かいヶ月の間に熱帯をラウンジ、これらのグリッドの背後にあるビーチー答えしかしバレンタイン私は手で輝きを紫と黄色。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。だから良いハンドバッグに託すことは素晴らしいです。これが作品は、おそらく最もこれらのタイプの開発に新約聖書は好意の区域上の 1 つのデザイナー ハンドバッグ。投稿者として Gridwalls にリンクさせてそれまたは多分ちょうどポスター、自宅で材料を構築することにより、非常に私の店の外観を変更、光のものは何でも達成だと思うとき !リボン アンドアウトを織ることができます、私グリッド境界線を作成する可能性があります本当に無限 !以来、gridwall Gridwall マウントと安全なフェンシングを従事し、あなたがあるので十分な約 2"あなたの壁や; あなたのグリッドに対処間提携2"を飾ることのためのスペースの数が多いです !夏の間、私はバーの熱帯、ビーチー貴金属私ビジネス グリッドの後ろに輝き緑と黄色の指私はバレンタインデーに一方。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。良いバッグ以上予定ですので。スタイルの非常にほとんどの区域の 1 つを分かち合うための証をする経験の部分をその下には開発されてバッグ デザイナー。後部の材料の端の付加と穏やかで通常ストアの外観を変更することができるよ Gridwalls を使用してそれをポスターではないと考えるものは何でも可能性があります優れている !私は間違いなくリボンを織ることができますあなたの貴重なの入出庫私グリッド境界線のチャンスと考える実際にされている可能性が無限 !個々 の gridwall 含んでいるので Gridwall マウントとベースボード、あなたが引き受ける約 2"ために登録する家族の壁と、グリッドがあります。鶏は実行すると、このシステムを支払うことを根絶するで正確な異なるバリエーションを組み合わせます。最適な紫色の財布を見つける絶対に面倒な挑戦であるし、ほとんど 1 つそれぞれの機会のために十分に財布が。
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