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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.
Posted by Christy Haynes on September 26, 2012 10:39 AM | Permalink
Minute Paper #4 – Sarah Anciaux 09/26/2012
Title: Improved spatial resolution in the imaging of biological tissue using desorption electrospray ionization
By: Cooks et. Al
Journal: Anal Bioanal Chem
In this paper the authors attempt to improve the spatial resolution seen with desorption electrospray ionization (DESI) in biological samples. The optimization of various parameters were performed on an biological sample of known morphology.
The authors developed a method to determine the spatial resolution of a DESI-MS by first preparing mouse brain and ovary tissue samples. They did this by collecting, freezing, and slicing the samples into 15 micron thick sections. The samples were thaw mounted onto general microscope slides and dried. With well-documented morphological features in the mouse samples, spatial resolution could be determined. Optimization parameters include solvent flow rate, solvent composition, scan-rate, scan time, step-size, and capillary diameter. After general optimization and resolution test of the DESI source by use of a red Sharpie, the authors moved on to the mouse brain. They found that by decreasing the step-size and scan speed while increasing the scan rate they improved the overall resolution. The authors were able to resolve 35 micron features compared to the previously reported value of 180 microns in biological samples. They also found that by changing the solvent composition for 1:1 water/methanol to 1:2 DMf/EtOH they were able to make the solvent more biologically compatible with the DESI source still functioning as desired.
The authors claim to have successfully developed a method to test the resolution of DESI-MS on biological samples while also increasing the resolution of the platform. There are some areas of this platform that I think could be addressed further. The authors claim that they have increased the resolution of DESI-MS in biological samples but they only looked at lipids over a very small m/z scan area. I question if they would still get the resolution they desire if they branch out to small proteins or even just small molecules. Also, the optimizations they made, while improving resolution, also increase the overall scan-time leading to longer analysis time. While this is bearable, it would be ideal to be able to optimize DESI-MS such that it can have improved resolution but also still have the short total analysis time that the platform is known for. I question if a smaller ID solvent capillary, creating a tighter analysis spot, as they described, might not be able to improve resolution if they were to also optimize other variables such as the stage to solvent spray tip and solvent spray tip to MS inlet parameters etc. Reducing the actual analysis spot size would allow for keeping the scan-rate faster and step size larger to decrease time but still maintain resolution if the smaller capillary solvent spray could be optimized. The final thing I think that could be addressed further is the preparation method of the samples. I think it would be useful to prepare the sample in a few different ways a see how resolution is affected by the condition of the tissue under investigation.
Sarah Anciaux |
September 26, 2012 11:53 AM
Minute Paper #4 (9/28/12) – Matt Irwin
Title: Catalytic Routes for Conversion of C1 Feedstocks
Speaker: A. Bhan
Seminar Date: 9/25/12: CEMS Seminar Series
The development of new methods to produce fuels and plastics from renewable resources is critical as the supply of nonrenewable resources continue to diminish. Although methods currently exist for the conversion of feedstock to methanol or methane, the subsequent use of these C1 reagents in reactions over inorganic catalysts to produce gasoline or other olefins is poorly understood and highly nonselective. To this end, the Bhan group is interested in developing a fundamental understanding of the underlying kinetics and reactions of C1feedstocks to higher products in order to selectively produce products of interest. Although the methods used were not given explicitly, they presumably include gas chromatography, 13C NMR, mass spectroscopy, infrared spectroscopy (IR), and x-ray absorption near edge structure (XANES) spectroscopy.
Dr. Bhan’s presentation was broken up into two sections: (1) the production of higher-order hydrocarbons from methanol and (2) coupling the dehydrogenation of methane to the deoxygenation of acetic acid for the efficient production of olefins. In the first half, Dr. Bhan described how the group used IR to quantitatively determine how methanol reacted with the surface hydroxyls on an inorganic catalyst at ~900 °C to yield surface methyls. A small fraction of the generated methyls were acidic, and these methyls were able to react with free ethylene introduced into the vapor to generate a 3-carbon intermediate which could then crack to form a free propene. This propene could then react with additional acidic methyls to generate further higher order hydrocarbons, thus outlining a step-wise addition process. It was also found that aromatics could similarly react with these surface acidic methyls to become substituted with methyls. In both cases, the higher order products often “shed” to form two lower order products which could subsequently react, forming two reaction pseudo-cycles. When both benzene and ethylene were added to the reactor, it was found that the this “shedding” process resulted in the aromatic reaction cycle feeding the olefin cycle and vice versa, resulting in two coupled reaction cycles. Thus, in order to promote the production of a particular product and monitor the reaction kinetics of one cycle, a cofeed was introduced. Ethylene was used to promote the formation of products from the aromatic cycle, while isobutane was used to suppress the aromatic cycle and thus promote the formation of products from the olefin cycle. Through thoughtful promotion of desirable reactions, the group was able to generate a product which was roughly 60% desirable hydrocarbons. The group also performed some limited work on performing dehydrogenation of methane and deoxygenation of acetic acid simultaneously. The motivation behind this work was to use the side product of the dehydrogenation as a reagent for the deoxygenation and vice versa in order to produce a more efficient process. Unfortunately, the group learned that such a coupling is not currently possible, as the catalyst used in the study is ultimately limited to a low product yield because the equilibrium thermodynamics are suppressed by the formation of hydrogen.
The work performed by the Bhan group provides great insight into reaction mechanisms which can utilize C1 feedstocks, but more work is necessary before such techniques can be broadly used. The methanol study currently is only able to yield 60% desirable products, and many of the side products generated are likely costly to separate due to their similar chemical composition. To remedy this issue, additional studies should be performed where different cofeeds are used to promote or suppress specific reactions within either of the reaction cycles. Beyond this, some of the product could be actively removed via a process such as distillation to promote reaction kinetics. Additionally, explicit rate expressions were not given for the individual reactions in the reaction cycles. Deducing these kinetics would allow for better determination of which reactions are rate-limiting or are most likely to form undesirable side products.
Matt Irwin |
September 26, 2012 3:00 PM
Title: Catalytic Routes for Conversion of C1 Feedstocks
Speaker: A. Bhan
Seminar Date: 09/25/12 CEMS Seminar Series
Prof. Aditya Bhan spoke about his group’s research regarding the use of zeolites for converting C1 feedstocks (such as methane and methanol) into longer hydrocarbons. Though zeolites are already commercialized, Bhan stated that the mechanisms of this catalytic strategy are not well understood. Therefore, the broad focus of his group’s efforts is to study the chemistry and reaction mechanisms of zeolite catalysis. Bhan shared the results from several of his students’ experiments, including titration, IR spectroscopy, x-ray absorption near edge structure (XANES) spectroscopy, and isotopic labeling experiments.
Dividing his seminar into two parts, Bhan first discussed the mechanistic studies of the conversion of methanol into longer hydrocarbons. He explained that methanol reacts with zeolite particles and forms methyl cations on the surface of the zeolite. The intermediates can then interact with an electron rich molecule, such as an olefin or an aromatic, to form a longer hydrocarbon. The resulting molecule can then continue to react with the surface methyls to form higher order hydrocarbons, such as iso-butylene or propylene. In order to control the selectivity of the reaction, different zeolites could be used. For instance, the zeolite H-SAO-34 is more selective towards producing light olefins while the zeolite H-ZSM-5 favorably produces branched olefins and alkanes. Though the selectivity differed, the Bhan group determined that the reaction mechanisms were identical among the catalysts by carrying out kinetic studies. These studies showed that for each of the zeolites the conversion of the methanol was first order in the olefin or aromatic feed. Therefore, the differences in the selectivity were due to the structure of the zeolite. Bhan argued that the steric and electrostatic interactions between the hydrocarbons and the zeolites were the rate determining factors.
For the second topic, Bhan detailed his group’s study of the production of fuel from methane, which is hydrogen rich, and biomass, which is hydrogen poor. The group first attempted this conversion by employing a co-catalyst strategy. They combined a MoO3 carbide, which induces both dehydration and deoxygenation reactions, with a zeolite, which would promote the step-wise growth of the hydrocarbon. The group determined that methane and acetic acid could be reacted to form benzene and hydrogen. However, because the reaction was reversible, the net reaction rate was severely hampered by the hydrogen pushing the reaction towards equilibrium. Additionally, the group discovered that the acetic acid actually oxidized the MoO3 carbide. By accounting for the oxidation in their theoretical models, the group determined the forward reaction rate, which is only due to the catalyst, was consistent with the results of the first topic.
Though Prof. Bhan’s seminar was excellent (he is a great speaker), the work he presented left room for some very interesting questions. For instance, though Bhan discussed the importance of zeolite structure in regards to selectivity, he did not detail how to tune the structure to produce the desired molecule. Additionally, he also did not discuss the catalytic mechanisms of the higher order hydrocarbon feeds, such as ethanol or propanol.
Ralm Ricarte |
September 26, 2012 10:28 PM
Minute Paper 4
Vibrational Spectroscopic Imaging and Multiphoton Microscopy of Spinal Cord Injury
Galli, R., et al
Journal Analytical chemistry
Injury to the spinal cord causes fibrotic scaring that hinders the axons from regenerating, breaking the vital connection our central nervous system has with the rest of the body. Therefore, it is important to understand what is happening to these cells and how different therapies affect them. Most methods to study this system involves staining the cells with different dyes or a form of immunhistochemistry in which you add several labeled antibodies, which attach to different parts of the tissue. In order to simplify the process, these authors used fourier transform infrared (FT-IR) and spontaneous Raman spectroscopy of non-stained spinal cord samples. They also stained samples to compare their results.
This study compared the spinal cord tissue samples from healthy Rats to ones with spinal cord injuries. The injury was caused when the researcher removed 2mm of spine from the left side between the 9th and 10th thorac. The covering tissue layers were kept intact and in place meaning the only thing different was the removed spine. After leaving the rats alone for three weeks, they were sacrificed and relieved of their spinal cords.
The FT-IR and Raman spectra were obtained and a spectral image looking at the clustering was made to compare it to the staining technique. They were able to show how each part of the spectra correlated with the different sections of the tissue including white matter, the lesion, the axons, macrophages, etc.
Since the spectra captured was able to determine the different sections and cells involved in the spinal cord, they compared the spectra of the non-injured to the injured spinal cord tissues. There were several noticeable differences in both FTIR and Raman. For FTIR, the injured tissue saw a decrease in the lipid content band intensities. The area of the lesion also showed the bands at 1110, 1280 and 1340cm-1 increase. Amide bands also decreased by the lesion area and collagen increased. For Raman, there was a substantially large increase in the intensity of the bands around 830-850 cm-1 for injured tissue. In order to look at the specific lipid content they used coherent anti-Stokes Raman scattering and SHG imaging to look at the collagen.
I think one the most important features looked at in this study for later studies is looking at the collagen increase around the injured tissue, showing that a fibrotic scar had formed. Surrounding this scar they could see the axons, but none went through it, which is more evidence that this collagen is forming a non-penetrable layer. Since it is important to look at how therapies can allow re-growth of axons, watching how collagen changes when axons grow could be beneficial. One direction the authors wanted to look at was in vivo experiment s, but this would be difficult with Raman due to the weak signals sent back, especially if the area is inflamed.
Sarah Gruba |
September 27, 2012 8:24 PM
Title: Layered Metal Sulfides Capture Uranium from Seawater
By: Manos et al.
Increasing global energy expenditure has initiated great demand for sustainable alternatives to fossil fuels. Although nuclear power may be a viable substitute for current energy sources, many questions concerning the environmental impact of long term nuclear fuel processing, usage and spills remain. Sulfide-based ion-exchangers (K2MnSn2S6 or KMS-1) demonstrated improved sequestering of UO22+ (uranyl ions) from potable, lake and seawater over current ion-exchange technologies. The methods included characterization of synthesized materials, as well as Mid-IR and solid-state NIR-UV-vis spectroscopy.
KMS-1 demonstrated potassium exchange with uranyl cations within the interlayer spacings of the material. IR spectra of the uranyl treated KMS-1 suggested substantial amounts of uranyl integration. This contrasted significantly with pristine KMS-1 measurements. Furthermore, NIR-UV-vis spectra revealed a sharp onset around 0.95 eV, which is likely due to charge transfer from a sulfur p orbital to a uranium 6d/5f orbital. Not only did this imply a high affinity of uranyl for the sulfide interface, it indicated uranyl might be redefined as a soft cation because of its interactions with soft ligands of KMS-1. Most importantly, KMS-1 demonstrated approximately ninety eight percent removal of uranyl from uranyl spiked potable, lake and seawater samples.
One striking feature of uranyl laden KMS-1 is its reversible nature in the presence of calcium carbonate (2 M, pH 10) without any structural deformations. Furthermore, uranyl integration into the sulfide interlayer spacing was accomplished over a wide range of pH values (3-9), which marked a significant improvement over other uranyl capture technologies. Lastly, KMS-1 sustained its uranyl selectivity despite treatment with high salt (CaCl2) concentrations. This is essential for developing seaworthy ion-exchange materials.
Although KMS-1 was demonstrated to be an effective uranyl removal material in aqueous environments, most of the work was conducted in small volumes. In order for KMS-1 to be a commercially viable option for uranyl removal in environmental systems, its affinity for uranyl must be maintained when the material is scaled up. Studies concerning the structural integrity of bulk KMS-1 should be conducted. One point of concern was the reversible nature of uranyl intercalating in KMS-1 by calcium carbonate solutions. How viable is KMS-1 as an uranyl sequestering material if its affinity for uranyl is reversed in alkaline solutions? This could significantly limit KMS-1 as an uranyl removal tool in lake and river systems. Work concerning modification of the KMS-1 sulfide interface should be aimed at making sulfide-uranyl interactions more “soft” and therefore less prone to disassociation in the presence of “hard” bases.
Sam Egger |
September 28, 2012 3:05 PM
Minute Paper 4 September 28, 2012
Title: FTIR Imaging of Brain Tissue Reveals Crystalline Creatine Deposits Are an ex Vivo Marker of Localized Ischemia during Murine Cerebral Malaria: General Implications for Disease Neurochemistry
By: Mark J. Hackett, Joonsup Lee and Fatima El-Assaad
Phosphocreatine is a molecule found in the skeletal muscle and brain formed from the amino acids glycine, methionine and arginine. It is a donator of high energy phosphate groups to ADP in order to create ATP, which cellular metabolism uses for energy. This process is imperative for proper cell functioning, and is even more of a necessity when impaired conditions threaten cell viability. Past research using Fourier transform infrared (FTIR) spectroscopic imaging found creatine micro deposits in situ using diseased brains of epileptic, amyloid lateral sclerosis and Alzheimer’s. The hypothesis was formed that these deposits served as possible disease specific markers or were a part of the brain pathogenesis. In this article, the authors challenge this hypothesis by studying cerebral malaria diseased mice once again with FTIR spectroscopic imaging.
FTIR spectroscopy operates by obtaining an absorption, emission or Raman scattering infrared spectrum of a sample over a wide spectral range collected simultaneously. The sample may be analyzed in a solid, liquid or gas state. For these experiments, an FTIR spectrometer was paired with a liquid-nitrogen-cooled focal plane array detector. FTIR imaging was the chosen technique because changes of the molecules’ distribution and thus concentration could be easily measured. The brains of diseased cerebral malaria mice were placed in optimal cutting temperature (OCT) medium for sample preparation. Samples were then frozen in liquid nitrogen cooled hexane and small portions of the cerebellum were removed and analyzed.
The FTIR spectroscopic imaging showed creatine micro deposits in situ and it was also observed that as dehydration (air-drying) of the post tissue cutting increased, the amount of deposits did as well. Since creatine deposits are soluble zwitterions and their amount increased during tissue processing, it was determined that their occurrence was due to creatine crystallization of brain tissue. This indicates that they have no effect as disease markers or on disease pathogenesis. It was further hypothesized that since decreased oxidative metabolism is prevalent in neurological disorders, this was the reason for observed high levels of phosphocreatine in past studies.
Although this study disproved an earlier hypothesis of observed phosphocreatine levels, it failed to distinguish between phosphocreatine micro deposits which accumulated due to poor oxidative metabolism and those which accumulated due to dehydration of brain tissues during the sample preparation. The authors acknowledge that micro deposits occur in vivo, but they proved that the large amount observed ex vivo was due to sample preparation. A good question to investigate would be at what point are the increased levels due to poor oxidative metabolism versus dehydration of tissue. In vivo experiments monitoring the time dependence of phosphocreatine deposits in neurological diseases accompanied by decreased oxidative metabolism would be able to determine whether these deposits are of value for further study of neurological disorders, or whether their abnormal occurrence is unrelated to any specific disease.
Megan Weisenberger |
September 28, 2012 3:29 PM
Minute Paper #3
Title: Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells
By: Craighead et.al.
In this paper the authors describe a microfluidic device designed to extract, purify, and unfold DNA from a single cell. This is accomplished by an array of micropillars in a channel to capture a single cell. Upon capture, the cell would then be exposed to a lysing agent, and DNA strands (Mbp in length) are caught while other reagents are washed away. The DNA strands can then be collected for further study.
Being able to isolate the DNA from one particular cell or small cell colony is very important in the area of Genomics. Microfluidic devices are desirable for this purpose since they require less reagent volumes, and they reduce the chance for human error with an enclosed system for separation. It can be particularly tricky to work with DNA in such small volumes as the microfluidic device operates on. Current methods use SPE for the extraction of DNA from small cell colonies (100 or fewer cells). They depend on the binding of DNA to silica or magnetic microparticles (BEAM). For these techniques, the DNA is loss is quite large since it is so sensitive to pH changes as well as other environmental factors. Overall the efficiency of this extraction is reported to be as low as 60% and as high as 90%. Which is sufficient for large colonies, but for smaller ones, or even one cell, we need a much better efficiency.
The authors then went ahead and created a liquid microfluidic device capable of separating and extracting DNA samples. It is made with PDMS, for flexibility and ease of production as well as keeping cost low. (I highly encourage you to look at the paper to see the picture of it so this will make more sense) Along the etched column, there are many micropillars designed to “catch” the DNA strands when the cell is destroyed. Toward the end, the pillars are randomized for maximum contact with DNA. From there, the leftovers of the cell are washed away, and then a restriction enzyme is used to digest the DNA. From there, the DNA is collected and sent away for further analysis and base calling. Figure 2 really shows this process well.
They then fluorescently labeled DNA strands and looked at how they were “caught” on the pillars, after one cell was introduced to the microfluidic. It works well. They were able to “essentially extract” 100% of genomic DNA from human chromosomes.
I wish the authors would elaborate more on what “essentially extract 100%” meant. Does that mean they don’t get it all? I also would like to see if this could be made into a one-pot technique. If you could do this, then amplify and analyze in one simple process that would be very useful. I’m not entirely sure how this could be accomplished.
Alex Johnson |
September 28, 2012 3:38 PM
Presentation Title: Catalytic Routes for Conversion of C1 Feedstocks
Speaker: Aditya Bhan
Seminar Date: 09/25/2012: CEMS Seminar Series
The conversion of the C1 hydrocarbons methane and methanol into more useful larger hydrocarbons is of great industrial and environmental interest. The presenter discussed the results of the combined effort of his group to elucidate the molecular mechanisms, kinetics, and selectivity for two processes capable of achieving C1 conversion: (1) the chain growth of olefins and methylation of aromatics by reacting methanol over zeolites known as methanol-to-hydrocarbons (MTH) and (2) the co-processing of hydrogen-rich methane and oxygen-rich, and thus hydrogen-deficient, biomass feedstock over bi-functional MoC/zeolite catalysts to produce useful fuels with minimum production of undesired H2 or CO. For the sake of brevity, I will discuss only the former project. The surface species present on the catalyst under reaction conditions was determined with transmission Fourier transform infrared spectroscopy (FTIR), while the composition of the reactor effluents was monitored with in-line mass spectrometry.
The O-H bands of the FTIR spectra of the zeolite catalyst were found to diminish when the catalyst was exposed to dimethyl ether (used as a gaseous substitute for methanol). Correspondingly, bands associated with the formation of methoxide species appeared upon exposure to dimethyl ether; demonstrating that, under reaction conditions, the active sites of the zeolite are almost completely covered by methoxide species which are capable of reacting with olefins and aromatics to induce methylation. The conclusion that the surface is saturated with methoxide species was supported by the observed zero-order kinetic dependence in methanol pressure for the observed products.
It was then proposed that the MTH process is carried out with the coupling of two catalytic cycles: (1) one in which surface methoxide species and olefins react to form larger olefins capable of either subsequently cracking into multiple smaller olefins or of cyclization into aromatic species and (2) one in which the produced aromatic species react with methoxide species to form more highly substituted aromatics capable of “shedding” to produce ethylene, thus feeding the other cycle. This view was supported by the observation that co-feeding either propylene or toluene changed the selectivities of the products by promoting the propagation of olefin and aromatic cycles, respectively; thus, allowing for the selective production of desired hydrocarbons.
While it was shown the methylation of aromatics and olefins in the MTH process occurs over a methoxide-saturated zeolite and the relative rates of propagation of the two catalytic cycles could be controlled by co-feeding olefins or aromatics, a complete mechanistic understanding of this process has yet to be acquired. By co-feeding 13C labeled aromatics and monitoring the resulting mass of the produced ethylene by using in-line mass spectrometry, one would be able to better understand how the shedding process occurs and determine whether the carbons in the ring play a catalytic role or whether ethylene is produced only by the interaction of the substituent groups of the aromatic with surface methoxide species. By investigating this, a better understanding of how these two cycles couple would be gained, which is a vital step in elucidating the reaction mechanism for this chemistry.
Joseph DeWilde |
September 28, 2012 3:50 PM
Title: Use of High Resolution Mass Spectrometry for Analysis of Polymeric Excipients in Drug Delivery Formulation
Author: Pilar Perez Hurtado
Journal: analytical chemistry
Increasing solubility of poor-water soluble drugs is one of the challenges in drug industry. Among large numbers of excipients which act as inert vehicles to solubilize the drugs and improve effective bioavailability, Polysorbate 80 and Gelucire 44/14 are two common choices for this purpose. In this paper, high resolution Fourier transform ion cyclotron resonance mass spectroscopy (FTICR MS) has been used to compare two batches of polysorbate 80 and Gelucire 44/14. All components of batches that have impact on drug delivery were analyzed and the differences between them were determined by employing the high resolution feature of the instrument.
Gelucire 44/14 is composed of mono-, difatty acid esters of PEG 1500, mono-,di, triglyceride, and free PEG. Polysorbate 80 is also made of isosorbide polyethoxylates PEG, polysorbate diesters and sorbitan polyethoxylates. Various instruments and methods have been already used to analyze these excipients such as IR, HPLC, TLC, GC, X-ray and MALDI-TOF MS. However, they could not differentiate some components because of their very close masses and their heterogeneity made their analysis more challenging. FTICR can easily detect similar masses with very small amount of errors by using cyclotron frequency of the ions in a stable magnetic field to measure the mass to charge ratio of ions.
In general, their mass analysis was based on distinguishing between the end group(s) from the repeat unit of polymers. The masses of end groups were determined by simply using masses of polymer, monomer, and adduct ion which is usually Fe+ or Na+.1
Two different samples (A and B) were employed and their mass spectra were recorded to investigate polymeric distribution pattern of Gelucire 44/14. Major components of the excipient were determined such as diester polymeric compounds with the mass errors of less than 0.630 ppm, but interestingly the signal for the PEG stearate as a constituent mentioned by the factory was not found. Also, three polymeric differences were detected by comparison of sample A and B. In PEG (H(OCH2CH2)nOH) were exist in sample A but not in B. The other differences were related to polyethylene polymer series that CH2CH2 repeat units and just found in sample A with the mass errors of -0.65 ppm.
Two samples (A and B) from two different companies were employed to determine components of polysorbate 80. Their major gradients were detected easily and accurately (very small specific mass errors), but no stearic acid was found in both of them.
The authors claimed that it’s the first time that FTICR instrument has been used to determine component of polysorbate 80 and Gelucire 44/14 and it’s capable of obtaining spectra with high resolutions. However, molecular structures of components cannot be elucidated by applying this method. MS/MS method can be used to study the structures and it can be performed with high resolution selection of the precursor fragments.
1-The authors has provided a few formulas in the paper which can be used to calculate end group(s) mass.
Marzieh Ramezani |
September 28, 2012 4:07 PM
Minute Paper #3
Tian Qiu 4651092
Title: Highly Sensitive Method for Assay of Drug-Induced Apoptosis Using Fluorescence Correlation Spectroscopy
Journal: Analytical Chemistry
Authors: Lingao Ruan, Jicun Ren, et al.
In this article, the authors presented a novel method using fluorescence correlation spectroscopy for the analysis of DNA fragmentation in apoptotic cells to detect the drug-induced apoptosis. Fluorescence correlation spectroscopy (FCS) was used to determine the fluctuation of the fluorescence intensity in a very small volume of sample. Because of the Brownian motion of the fluorophore-labeled DNA fragments in the solution, which means the number of the particles will randomly change around the average number, the fluorescence intensity will fluctuate, which can give out the average number of fluorescent DNA fragments and average diffusion time in the sample.
There have been several biochemical criteria to characterize apoptosis of cells, one of which is the fragmentation of chromosomal DNA. The fragmentation of DNA can be detected by gel electrophoresis or flow cytometry, etc. The authors chose the DNA fragmentation as the criteria, used FCS to achieve the high sensitive detection of the DNA fragmentations and also use flow cytometry and gel electrophoresis (DNA ladder assay) to make comparisons. SYBR Green I was used as fluorescent DNA-intercalating dye. In the FCS measurements, the single-component model and multiple-components model were both used to fit raw FCS data. The diffusion time was extracted from the data to show different cell status in apoptosis.
First they used standard DNA fragments to investigate the concentration effects of DNA and SYBR Green I and also the effects of different measurement time. The results showed there was no obvious change, which meant good reproducibility. The application of single-component model and multiple-component model was good with the correlation coefficients of 0.972-0.995 in the standard calibration. Then they used this FCS assay to detect drug-induced apoptosis under different concentrations of LDM. The results showed that the distribution of the characteristic diffusion times of DNA fragments significantly decreased with an increase in the concentrations of induced drug. At last, the result was compatible with the contrast assay of apoptosis by flow cytometer and gel electrophoresis.
I looked at those images and it seems there was not a very clear relationship between the sizes of the DNA fragments and the diffusion time. It was not linear. It seemed they could only perform a semi-qualitative assay. I’m hoping they could explore the possibility of performing quantitative assay in the future.
Tian Qiu |
September 28, 2012 4:08 PM
Title: Catalytic Routes for Conversion of C1 Feedstocks
Speaker: Aditya Bhan
Seminar date : 09/25/12 CEMS seminar
Professor Bhan presented his study about utilization of C1 source. He said renewable chemical sources would be important in the future for energy and other valuable chemicals. He mentioned methanol and methane as the important renewable sources. He said that these C1 sources could be changed to almost any chemicals by appropriate processes. He mentioned zeolite as a valuable catalyst that could catalyze C1 sources. Thus, he showed mechanistic studies of methanol and methane on zeolites. His group used gas chromatography (GC), mass spectroscopy(MS), infrared spectroscopy(IR), X-ray absorption near edge spectroscopy(XANES), and kinetic isotopic effects to study kinetics and mechanisms of C1 source reaction on zeolites.
His seminar could be divided to two parts; one for methanol, and the other for methane. For methanol, he said it is already widely used as a chemical source around the world using zeolite, process called methanol-to-hydrocarbon(MTH). However, he said there is no in-depth study why zeolites can convert methanol. He used IR to see how the zeolite surface interacts with methanol. Methanol interacts with hydroxyl group on zeolite and form CH3+ in the micropore of zeolite. His group could see the decrease in O-H peaks and the increase in C-H peaks in IR. This active CH3+ could react with either aromatics or olefins and also need those species to increase carbon number of chemicals. It cannot react itself to increase carbon number and need organic co-catalyst. The type of products is determined from the co-catalyst, aromatics or olefins. He showed the change of product selectivity according to added organics and confirmed that using kinetic labeling reactants. He also showed kinetics of those reactions and the reaction rate showed first order with added organics, which means the reaction with added organic species is rate-determining step. Thus, he could control the selectivity of products on zeolite using organic co-catalysts and methanol. For methane, he wanted to dehydrogenate methane by oxidation with oxygen-containing biomass. Then, methane is oxidized and biomass is reduced and hydrogen is produced. He wanted to couple these oxidation and reduction in one catalytic process. His group used molybdenium loaded zeolite for coupling reaction. They studied surface structure of Mo using XANES. When Mo was loaded, dimolybdenium oxide was formed. It is characterized by one shoulder peak in XANES. After carbon treating on the catalyst, dimolybdenium form disappeared. They made a reaction of methane with acetic acid, but, unfortunately, what they got was individual oxidation of acetic acid. It was not coupled in this catalyst and equilibrated quickly.
For methanol reaction, he studied quite well. However, even though he studied kinetics, he could study the elementary steps of each cycle, aromatic cycle and olefin cycle. If one can separate each elementary step, it would be easier to get target molecules. For this, he might be able to use computational modeling of interaction between zeolites and reaction intermediates. He could also check how organic co-catalysts are adsorbed on the surface using IR. For second topic, the result was different from what he intended. Anyway, I think the idea was good. I think he could check availability of the coupling reaction using computational method and find other oxygenates. He could also check how oxygenates interact with zeolite using IR, too.
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