NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) - 11.22.16
Scientists study chemical reactions using fluorescence polarization, which produces changes in light when molecules bind together. This technique enables researchers to measure the interactions of proteins with DNA or antibodies, and many other biomedical functions. NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) validates a commercial Plate Reader instrument that detects changes in light for these types of reactions in a multiwell plate, a flat plate with 384 wells or tiny test tubes, to examine microgravity’s effect on fluorescent polarization, which paves the way for advanced biology research and drug development in space. Science Results for Everyone
Information Pending Experiment Details
OpNom: NanoRacks Module-29
Siobhan Malany, Ph.D., Sanford Burnham Medical Research Institute, Orlando, FL, United States
Steve Vasile, Ph.D., Sanford Burnham Medical Research Institute, Orlando, FL, United States
NanoRacks LLC, Webster, TX, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
ISS Expedition Duration
September 2014 - March 2015; March 2016 - September 2016
- NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) validates the NanoRacks Plate Reader facility in three of its five modes of operation in order to examine the effect of microgravity on fluorescent polarization (FP) for a fluorophore in solution, to validate the UV-Visible mode by using three different absorbance wavelengths in serially diluted liquids, and to validate the fluorescence intensity by using serial dilutions of two different fluorophores. This investigation also serves as part of the process of validating and establishing a workflow for the NanoRacks Plate Reader facility. Testing is conducted with four microplate samples.
NanoRacks-Micro-gRx helps assess if molecular processes are the same in space as on Earth. This opens the door for future advanced biology and pharmacology research in microgravity. By transferring advanced technologies to the International Space Station (ISS), researchers are able to determine the effectiveness of medicines in microgravity and explore biochemical and cellular pathways that can be targeted for new disease therapies.
NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) experiments validate at least three of the five operating modes for the Molecular Devices Spectramax M5 multimode microtiter plate reader (NanoRacks Plate Reader), quantify the association of biological molecules as detected by fluorescent polarization (FP), and test the functionality of a 384-well microplate. These experiments are described by the following three aims:
Aim 1). Perform a fluorescent polarization experiment which validates the FP mode of the plate reader and provide experimental comparison for detecting the binding of molecules by FP in microgravity. FP measurement of a fluorescently labeled ligand binding to a larger molecule provides information on molecular orientation and mobility in solution of the bound complex versus the free ligand. An exact replica of the experiment is tested at the same time point on earth using the same instrument to detect changes in FP due to microgravity.
Aim 2). Validate the UV-Visible mode by using three different absorbance wavelengths in serially diluted liquids. Three absorbance wavelengths, which are the most commonly used by scientists, are tested.
Aim 3). Validate the fluorescence intensity mode by using serial dilutions of two different fluorophores (fluorescent chemical compounds that re-emit light upon light excitation). The two most commonly used fluorophores are tested during this portion of the M5’s validation.
These experiments are necessary to validate that the NanoRacks Plate Reader is operating correctly and to its specifications. The results generated from these experiments have interest to physicists, biophysicists, biologists as well as scientists working in the field of drug discovery who wish to use the plate reader for future life science experiments.
For the testing of the fluorescence polarization module of the NanoRacks Plate Reader, the use of a fluorescently labeled biotin conjugate and an antibody to biotin are employed. Biotin also known as vitamin H, coenzyme R or vitamin B7 is involved in the synthesis of fatty acids and glucose. This modified version of this molecule labeled with the fluorescent dye fluorescein is commercially available (see structure below).
Fluorescein-Biotin has an excitation wavelength maximum of 493 nm and an emission wavelength maximum of 518 nm. As this is a small molecule with a molecular weight of 831 Daltons, it tumbles rapidly in solution and thus, exhibits a low polarization value. In the presence of an antibody specific for biotin, the molecular volume (mw ≈ 150 kD) increases for the bound complex, thus causing tumbling of the bound fluorophore in solution to slow resulting in the complex having a high polarization value. NanoRacks-Micro-gRx serially dilutes the antibody while keeping the fluorescein-biotin concentration constant as in the experiment shown above. The investigation starts with a concentration of antibody which has all of the fluorescein-biotin bound and go down to no antibody (all fluorescein-biotin is unbound). As the antibody becomes more and more limiting the amount of free fluorescein-biotin in solution increases, which in turn causes the FP value to decrease. Serial dilutions of the antibody are made in sterile phosphate buffered saline (PBS) pH 7.2, with a single dilution added to an individual well of a 384 well black microtiter plate. Every well that contains the antibody solution also receives an equal amount of fluorescein-biotin. As they use the same color plate, the FP experiment is on the same plate as the fluorescence intensity experiment. A diagram of the experiment is shown in the fluorescence intensity section below.
Four identical plates using the same stock reagents are created prior to launch. Two are loaded into NanoRacks Module-29 while the others are kept at Sanford Burnham Medical Research Institute. For a 384 well plate approximately 30 µl of solution is in each sample well. Once the reagents have been added to the wells the plate is sealed with an optically clear seal.
For the testing of the Fluorescence intensity module of the plate reader, the use of two different fluorescent dyes is employed. The two dyes are a fluorescein derivative and a rhodamine derivative. Fluorescein has an excitation wavelength of 490 nm and emission, while rhodamine excites at around 540 nm and emits at around 570 nm. These wavelengths change slightly depending on the derivative that is finally chosen.
Serial dilutions of each fluorophore are made in sterile phosphate buffered saline (PBS) pH 7.2, with a single dilution added to an individual well of a 384 well black microtiter plate. They are placed on the same plates as the FP experiment.
For the testing of the UV-Visible Absorbance module of the NanoRacks Plate Reader, the use of three different food colors found at one’s local supermarket is employed. We anticipate using a yellow dye (absorbance wavelength of ~420 nm) a red dye (absorbance wavelength of ~540 nm) and a green dye (absorbance maximum of ~620 nm). Prior to launch the absorbance spectra of each of the food colors is measured to determine the maximum absorbance wavelength for each of the dyes.
Serial dilutions of each dye are made in water, with a single dilution added to an individual well of a 384 well clear microtiter plate. Approximately 30 ul of solution is added to each well. Once the serial dilutions have been added to the wells the plate is sealed with an optically clear seal.
The ways in which certain molecules change in response to light can tell scientists something about their orientation, movement and interactions with other molecules. Scientists use this technique, called fluorescence anisotropy, to study new drugs to treat disease. But many molecules behave differently in space than they do on Earth. This investigation studies how microgravity affects molecules and the process of fluorescence anisotropy. Understanding differences between this technique in microgravity and on Earth will help researchers study new medicines and explore new cell therapies in space.
Molecules and cells may behave differently in space, allowing researchers to perform unique experiments and to study biology in new ways. Results from this investigation open the door for future advanced research in biology and pharmacology, or the study of drugs, in microgravity. Developing new drugs in space may lead to new treatments for a wide range of human diseases, benefiting people on Earth.
Operational Requirements and Protocols
NanoRacks Module-29 transfers from +4°C Cold-Stow to ISS ambient conditions and assays are run on the 4 different types of Microplates with the NanoRacks Plate Reader facility by ISS Docking + 5 days. The 6 USB flashdrives are utilized to load the assay protocol files onto the NanoRacks Plate Reader for the analyses of the Microplates. Once data is downlinked for analysis and operations are complete (per Payload Developer confirmation) the NanoRacks Module-29, Microplates, and USB flashdrives are discarded.^ back to top
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NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) validates the NanoRacks Plate Reader facility in three of its five modes of operation. Image courtesy of NanoRacks LLC.
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Example of M5 Ground Unit used in NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx) to assess differences in spectroscopic analysis between ground and the NanoRacks Plate Reader facility aboard the ISS. Image courtesy of Molecular Devices, LLC.
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For absorbance measurements a flat bottom natural polypropylene 384-well microplate is used in NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx). Image courtesy of Greiner Bio-One.
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For fluorescence measurements a flat bottom black non-binding 384- microplate is used in NanoRacks-Fluorescent Polarization in Microgravity (NanoRacks-Micro-gRx). Image courtesy of Greiner Bio-One.
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