Robotic arm probes chemistry of 3-D objects by mass spectrometry

When life on Earth was first getting started, simple molecules bonded together into the precursors of modern genetic material. A catalyst would have been needed, but enzymes had not yet evolved. One theory is that the catalytic minerals on a meteorite's surface could have jump-started life's first chemical reactions. But scientists need a way to directly analyze these rough, irregularly shaped surfaces. A new robotic system at Georgia Tech's Center for Chemical Evolution could soon let scientists better simulate and analyze the chemical reactions of early Earth on the surface of real rocks to further test this theory.

In a proof-of-concept study, scientists selected a region for analysis on round or irregularly-shaped objects using a 3-D camera on a robotic arm, which mapped the 3-dimentional coordinates of the sample's surface. The scientists programmed the robotic arm to poke the sample with an acupuncture needle. The needle collected a small amount of material that the robot deposited in a nearby mass spectrometer, which is a powerful tool for determining a substance's chemical composition.

"You see the object on a monitor and then you can point and click and take a sample from a particular spot and the robot will go there," said Facundo Fernandez, a professor in the School of Chemistry and Biochemistry, whose lab led the study. "We're using an acupuncture needle that will touch very carefully on the surface of the object and then the robot will turn around and put the material inside of a high resolution mass spectrometer."

The research was published online February 28 in the journal Analyst, a publication of the Royal Society of Chemistry. The work was supported by a National Science Foundation (NSF) Major Research Instrumentation Program (MRI) grant and by the National Science Foundation (NSF) and NASA Astrobiology Program, under the NSF Center for Chemical Evolution.

Mass spectrometry is a powerful tool for analyzing surface chemistry or for identifying biological samples. It's widely used in research labs across many disciplines, but samples for analysis typically have to be cleaned, carefully prepared, and in the case of rocks, cut into thin, flat samples. The new robotic system is the first report of a 3-D mass spectrometry native surface imaging experiment.

"Other people have used an acupuncture needle to poke a sample and then put that in mass spec, but nobody has tried to do a systematic, three-dimensional surface experiment," Fernandez said. "We are trying to push the limits."

To show that the system was capable of probing a three-dimensional object, the researchers imprinted ink patterns on the surfaces of polystyrene spheres. The team then used the robotic arm to model the surfaces, probe specific regions, and see if samples collected were sufficient for mass spectrometry analysis. The researchers were able to detect inks of different colors and create a 3-D image of the object with sufficient sensitivity for their proof-of-principle setup, Fernandez said.

The research was the result of collaboration between Fernandez's group, which specializes in mass spectrometry, and Henrik Christensen's robotics group in the College of Computing. Christensen is the KUKA Chair of Robotics and a Distinguished Professor of Computing. He is also the executive director of the Institute for Robotics and Intelligent Machines (IRIM) at Georgia Tech.

"The initial findings of this study mark a significant step toward using robots for three-dimensional surface experiments on geological material," Christensen said. "We are using the repeatability and accuracy of robots to achieve new capabilities that have numerous applications in biomedical areas such as dermatology."

"It doesn't happen very often that a group in mass spectrometry will have a very talented robotics group next to them," Fernandez said. "If we tried to learn the robotics on our own it could take us a decade, but for them it's something that's not that difficult."

Christensen's team loaned a Kuka KR5 sixx R650 robot to Fernandez's lab for the study. Afterwards, Fernandez's lab purchased their own robot from Universal Robots. They have also upgraded to a new mass spectrometer capable of resolution nearly eight times higher than the one used in the study. They will soon begin replicating early Earth chemistry on rocks and analyzing the reaction products with their robotic sampling system.

"We really want to look at rocks," Fernandez said. "We want to do reactions on rocks and granites and meteorites and then see what can be produced on the surface."

The technology could also be applied to other research fields, Fernandez said. For example, the robot-mass spec combo might be useful to dermatologists who often probe lesions on the skin, which have distinct molecular signatures depending on if the lesion is a tumor or normal skin tissue.

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The above story is based on materials provided by Georgia Institute of Technology. Note: Materials may be edited for content and length.

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Clever chemistry improves new class of antibiotics

Jan. 17, 2014 — As concerns about bacterial resistance to antibiotics grow, researchers are racing to find new kinds of drugs to replace ones that are no longer effective. One promising new class of molecules called acyldepsipeptides -- ADEPs -- kills bacteria in a way that no marketed antibacterial drug does -- by altering the pathway through which cells rid themselves of harmful proteins.

Now, researchers from Brown University and the Massachusetts Institute of Technology have shown that giving the ADEPs more backbone can dramatically increase their biological potency. By modifying the structure of the ADEPs in ways that make them more rigid, the team prepared new ADEP analogs that are up to 1,200 times more potent than the naturally occurring molecule.

A paper describing the research was released on-line by the Journal of the American Chemical Society.

"The work is significant because we have outlined and validated a strategy for the enhancing the potency of this promising class of antibacterial drug leads," said Jason Sello, professor of chemistry at Brown and the paper's senior author. "The molecules that we have synthesized are among the most potent antibacterial agents ever reported in the literature."

"The molecules that we have synthesized are among the most potent antibacterial agents ever reported in the literature."ADEPs kill bacteria by a mechanism by that is distinct from all clinically available anti-bacterial drugs. They work by binding to a protein in bacterial cells that acts as a "cellular garbage disposal," as Sello describes it. This barrel-shaped protein, called ClpP, breaks down proteins that are misfolded or damaged and could be harmful to the cell. However, when ClpP is bound by an ADEP, it's no longer so selective about the proteins it degrades In essence, the binding by ADEP causes the garbage disposal to run amok and devour healthy proteins throughout the cell. For bacteria, a runaway ClpP is deadly.

ADEPs have been shown to kill bacteria that cause staph infections, some kinds of pneumonia, tuberculosis, and other types of infection in the lab. The molecules have also been reported to cure bacterial infections in mice and rats.

ADEPs were first discovered as naturally occurring compounds. Certain bacteria produce them for chemical defense. But for the last few years, scientists including Sello's group have been making synthetic ADEP analogs, in the hope of identifying compounds with potential as new drugs.

One approach the researchers thought might work involves making the ADEP molecule more rigid. Compared to the ClpP molecule to which it binds, the ADEP molecule is a bit "floppy," Sello said. "We often use the expression 'lock and key' to describe how a small molecule binds to a protein. One can imagine that it is easier to fit a rigid key into a lock rather than a floppy key. In the same sense, rigid molecules often bind to their protein targets more tightly."

Sello and his team synthesized several new ADEP molecules. They swapped out certain amino acids in the naturally occurring molecule with ones they thought might increase the molecule's rigidity. To find out if the new molecules were indeed more rigid, the team performed experiments that tested the strength of hydrogen bonds within the molecule. Stronger hydrogen bonds would indicate a more rigid molecule.

The researchers placed ADEP molecules in a solution rich in deuterium, a hydrogen atom that has an extra neutron. Over time, the deuterium atoms in the solution will swap places with the hydrogen atoms in the ADEP molecules. The deuterium swap happens more slowly, however, when hydrogen atoms are involved with strong bonds. So if the modified ADEPs exchanged deuterium more slowly, it would be an indication of strong bonds and a more rigid molecule.

The experiments showed that the modified ADEPs exchanged deuterium as much as 380 times more slowly than the natural molecule, a clear indication that the molecules were more rigid.

"It was exciting to see how rather simple modifications to the ADEP structure could affect their rigidity in such a profound manner," said Daniel Carney, a graduate student in Sello's group. "More importantly, the results were in line with our ADEP design principle. It is always rewarding when a sophisticated chemical theory can be applied and validated by laboratory experiments."

To follow up on the prediction that the rigid ADEPs would bind ClpP more tightly, Robert Sauer and Karl Schmitz at MIT measured the capacity of the ADEP analogs and the parent compound to produce the "runaway garbage disposal" phenomenon in solutions containing the ClpP protein. The experiments showed that the modified ADEPs produced the effect at much lower concentrations, indicating a higher binding efficiency. The results implied that the modified molecules were about seven times better than the standard ones at binding to ClpP.

The final step was testing whether the rigid ADEPs were better at killing bacteria in a test tube. Those tests showed that, compared to published reports for standard ADEPs, the modified compounds were much more potent against three different dangerous bacteria -- 32 times more potent against S. aureus, 600 times more potent against E. faecalis, and 1,200 times more potent against S. pneumoniae.

Sello was a bit surprised by the dramatic increase in ADEP potency compared to the much more modest improvement in ClpP binding.

"We found that the most potent ADEP analog binds ClpP seven-fold better than the parent compound, yet it has 1,200-fold better antibacterial activity," Sello said. "We believe that some of the increase in potency may stem from the fact that the rigidified ADEPs bind ClpP more tightly and have an enhanced capacity to cross the cell membrane. The improved cell permeability of the ADEP analogs is consistent with reports in the literature that molecules with strong intramolecular hydrogen bonds are particularly good at penetrating cells."

Sello and his team are encouraged by their results and are working to develop the ADEPs into next-generation antibacterial drugs. The next step -- a study to test how well the compounds work in mice -- is already underway.

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