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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.

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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
http://pubs.acs.org/doi/abs/10.1021/ac3025202
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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