Scientists devise new imaging technique for analysis of biological samples

"The beauty of the technique is that it doesn't require any sample preparation," said Dr. Julia Laskin, who led the research project for PNNL's Chemical Imaging Initiative. "Here, you just take your sample, slice it, and put it in front of the instrument for analysis."

The technique, known as Nanospray Desorption Electrospray Iionization or nano-DESI, allows scientists to efficiently determine which molecules reside in a precise spot on a sample. With this information, researchers can learn more about how diverse biological samples, such as tissues and microbes,  respond to environmental factors. For example, biochemists can gain see how the marine microbe Shewanella oneidensis alter metals to remediate hazardous materials in the soil. For example, S. oneidensis can change very soluble hexavalent uranium to less soluble form, limiting its movement in groundwater. Another opportunity lies with medical researchers learning how nicotine and other toxins affect brain cells.

"Great discoveries often require great tools," said Dr. Louis Terminello, who leads the Chemical Imaging Initiative at PNNL. "The discoveries needed to solve today's problems aren't something that you're going to get by eyeballing a sample."

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This sample shows an overlay of the optical and nano-DESI image of a human kidney tissue sample.

From the beginning, the team was convinced that the liquid bridge used in nano-DESI could be scaled down to analyze small areas on biological samples. By making adjustments, the team was able to scale the probe down to analyze an area about 10 micrometers in diameter, about the same size as a single red blood cell or mid-sized bacteria.

"With this probe, we are getting down to individual cells," said Laskin.

The team first analyzed rat brain tissues, which provide an outstanding test case for the technique, because they are very dense and yield high signals. The nano-DESI was able to draw up and analyze the molecules from different regions on the sample. Then, the team moved onto "airy" kidney tissues, and again were able to analyze micrometer-sized areas.

With each sample, the nanoDESI generated reams of mass spectra. Existing software packages could not process the data. The job fell to Brandi Heath, a team member who recently completed her bachelor's degree and is working on her master's degree at Washington State University. She read the charts and determined the fatty acids, amino acids, lipids, and other molecules that resided at different locations on the tissue.

"It was very tedious, but in the end, very rewarding," said Heath.

"Compared to other online liquid extraction techniques, nano-DESI has about an order of magnitude better spatial resolution," Laskin added. "It is comparable, spatially, to what laser-based techniques can give."

The team is working on two efforts related to their work with nanoDESI. First, they are working with Drs. Dongsheng Li and James Carson through the Chemical Imaging Initiative to understand and visualize the mass spectrometry data on the fly. Also, the team is working with Drs. Matthew Marshall, Margaret Romine, Grigoriy Pinchuk, and Jim Fredrickson to analyze different microbial communities of interest to the Department of Energy.

More information: Laskin J, et al. 2012. "Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry." Analytical Chemistry 84(1):141-148. DOI: 10.1021/ac2021322

Provided by Pacific Northwest National Laboratory (news : web)

Getting to xenon: Scientists examine alternatives for pulling this rare, expensive element out of air samples

"The promise of NiDOBDC and similar metal-organic frameworks is that we will be able to make faster and more sensitive separation and monitoring equipment," said Grate, who has been working in materials and systems for sensing and nonproliferation applications for his entire career at PNNL.

Absorbents, like activated charcoal and MOFs, can selectively capture xenon and release it on demand. Making these absorbents more efficient could benefit sustainable processes and national security. Commercial uses of xenon include lighting, scientific instruments, and anesthesia. Security uses focus on nuclear processes. Nuclear reprocessing, weapons tests, and nuclear accidents, such as the 2011 catastrophe in Japan, release xenon radioisotopes into the atmosphere. Around the world, monitors track xenon for the Comprehensive Test Ban Treaty.

The research team compared the performance of three materials that capture xenon. Conventional technologies for capturing xenon use activated charcoal, which is fine black carbon powder processed to be porous, with an effective surface area of 500 square meters a gram. Charcoal also absorbs xenon from air samples and releases it on demand.

"Activated charcoal is a really old-fashioned material with little potential for improvement," said Grate. "We wanted to see if we could find a material that we could push, that we could get new capabilities from."

Metal-organic frameworks or MOFs were an obvious starting point. NiDOBDC, MOF-5, and other members of this new class of framework-based sorbents are extremely porous. The frameworks can achieve surface areas ~10 times greater than activated charcoal. Further, the modular nature of the MOF synthesis method and their internal chemistry lets scientists build materials with a range of structures and properties.

"Interest in MOFs has exploded in the past decade," said Thallapally, who is also conducting research for DOE's Office of Nuclear Energy using these same materials.

At PNNL, Thallapally was working with MOFs for carbon sequestration, but saw more potential for the materials. He met with Grate to discuss MOF possibilities for security applications. They believed PNNL had the right capabilities to synthesize and characterize the necessary materials. They applied for and received PNNL funding to examine how well MOFs worked in xenon capture.

Previously, some MOFs proved disappointing. The materials worked well in the lab, but their framework structures were unstable under environmental conditions as vapors were sorbed and desorbed.

Working in PNNL's Sigma 5, the researchers tested three different xenon absorbers: NiDOBDC, activated charcoal, and a prototypical MOF, known as MOF-5. They found NiDOBDC takes up xenon about as well as activated charcoal and significantly better than MOF-5. NiDOBDC is superior at lower pressures. At 1 bar, the pressure at sea level, NiDOBDC was able to take up significant amounts of xenon and release all of it when the conditions were right. They also found that NiDOBDC was more selective than charcoal for xenon over krypton, which is fairly similar to xenon.

Thallapally and Grate are continuing to investigate MOFs, including NiDOBDC. Their work will include studies on scaling the materials to study their performance in real gas streams.

"While there isn't much you can do to improve charcoal, there is a world of potential in MOFs," said Thallapally. "The defined nanostructured framework gives you a lot of opportunities synthetically to add more functionality."

More information: PK Thallapally, et al. 2011. "Facile Xenon Capture and Release at Room Temperature using a Metal-Organic Framework: A Comparison with Activated Charcoal." Chemical Communications. DOI: 10.1039/C1CC14685H

Provided by Pacific Northwest National Laboratory (news : web)

Researchers expand capabilities of miniature analyzer for complex samples

It’s not often that someone can claim that going from a positive to a negative is a step forward, but that’s the case for a team of scientists from the National Institute of Standards and Technology (NIST) and private industry. In a recent paper,* the group significantly extended the reach of their novel microfluidic system for analyzing the chemical components of complex samples. The new work shows how the system, meant to analyze real-world, crude mixtures such as dirt or whole blood, can work for negatively charged components as well as it has in the past for positively charged ones.

In previous work,** NIST researchers Elizabeth Strychalski and David Ross, in collaboration with Alyssa Henry of Applied Research Associates Inc. (Alexandria, Va.), demonstrated the use of a technique called GEMBE (for “gradient elution moving boundary electrophoresis”) for analyzing complex samples. The NIST-developed system combines a simple microfluidic structure (two reservoirs connected by a microchannel), electrophoresis (which uses electricity to move sample components through a fluid) and pressure-driven flow.

Analyzing complex samples can be difficult because components in these samples (such as the fat globules in milk or proteins in blood) can “foul” or contaminate microfluidic channels. The traditional solution has been to remove contaminants with costly, time-consuming sample preparation prior to analysis.

GEMBE solves this problem by pumping fluid through the microchannel using a controlled pressure in the direction opposite to electrophoresis. This opposing pressure-driven flow acts as a "fluid gate" between the sample reservoir and the microchannel. Gradually reducing the pressure of the counterflow opens the "gate" a little bit at a time. A specific sample component is detected when the pressure flow becomes weak enough—i.e. the "gate" opens wide enough—that the component’s electrophoretic motion pushes it against the pressure-driven flow and into the channel for detection. In this way, different components enter the channel at different times, based on their particular electrophoretic motion. Most importantly, the channel doesn’t become fouled because the unwanted components in the sample are held out.

“Previously, we validated the GEMBE technique by quantitatively analyzing components from complex samples in solution that were cationic [positively charged] and could, therefore, be separated relatively easily from anionic [negatively charged] contaminants in a mixture,” Strychalski says. “However, we needed a way to make GEMBE work when both the desired components and the contaminants are negatively charged.”

For some samples, Strychalski says, this was achieved by choosing a different solution pH to change the electrophoretic motion of the unwanted components. In other cases, the addition of commercially available surface coatings to the sample did the trick without compromising the ease and robustness of the GEMBE technique.

“Additives can be selected that will interact with material in the sample that we don’t want to study,” Strychalski explains. “If we choose the right coating, it will slow the electrophoretic motion of contaminants relative to the desired components. This prevents the former from interfering with analysis while still allowing the latter to enter the microchannel for detection.”

Strychalski and her colleagues plan to continue refining the GEMBE system, including an effort to define which surface coatings optimize the technique for specific components in a variety of complex samples.

More information: E.A. Strychalski, et al. Expanding the capabilities of microfluidic gradient elution moving boundary electrophoresis for complex systems. Analytical Chemistry, Vol. 83, No. 16, pp 6316–6322. Aug. 15, 2011.

See “‘No Muss, No Fuss’ Miniaturized Analysis for Complex Samples Developed” http://www.physorg … 7763391.html

Provided by National Institute of Standards and Technology (news : web)

Chemist develops biosensor that changes color when bacteria are present in water samples

A team of chemists led by Vincent M. Rotello of the University of Massachusetts Amherst has developed a fast, simple and low-cost field test for detecting bacteria in low concentrations in drinking water using a biosensor made of gold nanoparticles, an enzyme and dye. The biosensor can detect harmful bacteria in concentrations as low as 100 cells per milliliter. Their report appears in the current online edition of the Journal of the American Chemical Society.

The new test could have a significant impact in developing countries where public health workers, physicians and water quality specialists are most in need of a quick, sensitive way to detect pathogens such as bacteria in a water supply. The time it takes to culture samples and wait for relatively expensive lab results severely hampers efforts to save the estimated 300 million people affected by bacterial illness each year. Estimates are that more than 2 million children die annually from bacteria-related disease.

Currently, there are many methods, some quite sophisticated for detecting harmful bacteria such as the killer E. coli. These include culturing, nucleic acid probes and microarrays. But clinics and environmental managers in developing nations often don’t have access to them because of high cost or the need for skilled technicians to read the results.

To address this problem, the research team headed by Rotello with partners at the University of Puerto Rico and the Georgia Institute of Technology, developed a test strip suitable for field use that has a simple visual read out. This new uses enzyme-nanoparticle assemblies absorbed on paper strips. When the paper comes in contact with bacteria, the enzyme is activated and the strip turns from yellow to red, an easily observable change that takes place within 10 minutes.

Rotello also notes that very small amounts of the nanoparticles and enzyme are needed for the reaction, keeping the price of the test strips low. The team is now working to improve the sensitivity of the test strips to be able to detect even smaller amounts of bacteria.

The work is supported by the National Science Foundation through the UMass Amherst Center for Hierarchical Manufacturing.

Provided by University of Massachusetts Amherst (news : web)

'Lost' samples from famous origin of life researcher could send search for first life in new direction

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Preserved samples from a 1958 experiment done by "primordial soup" pioneer Stanley Miller contain amino acids created by the experiment. The samples had not undergone analysis until recently when Miller's former student Jeffrey Bada and colleagues discovered a wide range of amino acids. The find could be an important step toward understanding how life on Earth could have originated. The vials have been relabeled but the boxes are marked with Miller's original notes. Credit: Scripps Institution of Oceanography, UC San Diego

(PhysOrg.com) -- Stanley Miller gained fame with his 1953 experiment showing the synthesis of organic compounds thought to be important in setting the origin of life in motion. Five years later, he produced samples from a similar experiment, shelved them and, as far as friends and colleagues know, never returned to them in his lifetime.

More 50 years later, Jeffrey Bada, Miller's former student and a current Scripps Institution of Oceanography, UC San Diego professor of marine chemistry, discovered the samples in Miller's laboratory material and made a discovery that represents a potential breakthrough in the search for the processes that created Earth's first life forms.

Former Scripps undergraduate student Eric Parker, Bada and colleagues report on their reanalysis of the samples in the March 21 issue of Proceedings of the National Academy of Sciences. Miller's 1958 experiment in which the gas hydrogen sulfide was added to a mix of gases believed to be present in the atmosphere of early Earth resulted in the synthesis of sulfur amino acids as well as other amino acids. The analysis by Bada's lab using techniques not available to Miller suggests that a diversity of organic compounds existed on early planet Earth to an extent scientists had not previously realized.

 

Scripps Oceanography professor of Marine Chemistry Jeffrey Bada holds a preserved sample from a 1958 experiment done by "primordial soup" pioneer Stanley Miller. The residue in the sample contains amino acids created by the experiment. The samples had not undergone analysis until recently when Bada and colleagues discovered a wide range of amino acids using modern detection methods. Credit: Scripps Institution of Oceanography, UC San Diego

The new findings support the case that volcanoes — a major source of atmospheric hydrogen sulfide today — accompanied by lightning converted simple gases into a wide array of amino acids, which are were in turn available for assembly into early proteins.

Bada also found that the amino acids produced in Miller's experiment with hydrogen sulfide are similar to those found in meteorites. This supports a widely-held hypothesis that processes such as the ones in the laboratory experiments provide a model of how organic material needed for the origin of life are likely widespread in the universe and thus may provide the extraterrestrial seeds of life elsewhere.

Successful creation of the sulfur-rich amino acids would take place in the labs of several researchers, including Miller himself, but not until the 1970s.

"Unbeknownst to him, he'd already done it in 1958," said Bada.

Miller's initial experiments in the 1950s with colleague Harold Urey used a mixture of gases such as methane, ammonia, water vapor and hydrogen and electrically charged them as lightning would. The experiment, which took place in a closed chamber meant to simulate conditions on early Earth, generated several simple amino acids and other organic compounds in what became known as "primordial soup."

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This is a photo of Stanley Miller in his UC San Diego lab in 1970. Credit: Scripps Institution of Oceanography Archives

With the gases and electrical energy they produce, many geoscientists believe the volcanoes on a young planet covered much more extensively by water than today's served as oases of raw materials that allowed prebiotic matter to accumulate in sufficient quantities to assemble into more complex material and eventually into primitive life itself. Bada had already begun reanalyzing Miller's preserved samples and drawing conclusions about the role of volcanoes in sparking early life when he came across the previously unknown samples. In a 2008 analysis of samples left from Miller's more famous experiment, Bada's team had been able to detect many more amino acids than his former mentor had thanks to modern techniques unavailable to Miller.

Miller, who became a chemistry professor at UCSD in 1960, conducted the experiments while a faculty member at Columbia University. He had collected and catalogued samples from the hydrogen sulfide mix but never analyzed them. He only casually mentioned their existence late in his life and the importance of the samples was only realized shortly before his death in 2007, Bada said. It turned out, however, that his 1958 mix more closely resembled what geoscientists now consider early Earth conditions than did the gases in his more famous previous experiment.

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The original box containing archived spark discharge samples prepared by Stanley Miller in 1958. For unknown reasons, Miller never analyzed these even though this is his first experiment using hydrogen sulfide. The label shows Miller?s original writing: p 114 refers to his notebook. Credit: Jeffrey Bada and Robert Benson/Scripps Institution of Oceanography, University of California at San Diego

"This really not only enhances our 2008 study but goes further to show the diversity of compounds that can be produced with a certain gas mixture," Bada said.

The Bada lab is gearing up to repeat Miller's classic experiments later this year. With modern equipment including a miniaturized microwave spark apparatus, experiments that took the elder researcher weeks to carry out could be completed in a day, Bada said.

Provided by University of California - San Diego (news : web)