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DOE OSTI2021

The fluorescence quantum yield parameter in Förster resonance energy transfer (FRET)—Meaning, misperception, and molecular design

The fluorescence quantum yield parameter in Förster resonance energy transfer (FRET) processes underpins vital phenomena ranging from light harvesting in photosynthesis to design of sensors for monitoring physiological processes. The criteria for choosing a donor for use in FRET processes include chemical features (solubility, bioconjugatability, synthetic accessibility, and stability) as well as photophysical properties pertaining to absorption (wavelength and molar absorption coefficient) and fluorescence (wavelength and fluorescence quantum yield). The value of the donor fluorescence quantum yield (Φf, or emphatically, Φf(D)) alone has sometimes been thought (erroneously) to place a ceiling on the possible quantum yield of energy transfer (Φtrans). A high value of the donor Φf, while attractive, is not at all essential; indeed, many valuable candidates for use as FRET donors have likely been excluded on the basis of this injudiciously applied filter. Such disregard is unwarranted. In this tutorial overview, the equations for FRET are reviewed along with pertinent core concepts in photophysics. An analogy using simple hydraulics provides a pedagogical tool for the non-aficionado to better understand photochemical kinetics. Ten examples are presented of donor–acceptor systems with donors that exhibit a range of Φf values (0.60, 0.59. 0.21, 0.17, 0.12, 0.118, 0.04, 0.018, 0.007, and 0.003; i.e., 60%–0.3%), yet for each corresponding donor–acceptor pair, the value of Φtrans is at least 0.70 and in some cases nearly 1.00 (i.e., 70%–100%). The systems encompass protein, synthetic inorganic, and synthetic organic architectures. The objectives of this illustrative review are to deepen understanding of FRET and to broaden molecular design considerations by enabling selection from among a far richer set of donors for use in FRET processes.

Lindsey, Jonathan S. (ORCID:0000000248722040)↗

NASA NTRS1975

Fretting of titanium at temperatures to 650 C in air

Fretting wear experiments were conducted on high-purity titanium at temperatures up to 650 C. Results indicate that up to about 500 C, the fretting wear increases with temperature. A further increase in the temperature up to 650 C results in decreasing fretting wear. This change in trend of fretting wear with temperature is shown to be associated with a change in oxidation rate. Additional experiments at 650 C showed a transmission from a low rate of fretting wear to a higher rate occurred after exposure to a number of fretting cycles; the number of cycles required to cause this transition was dependent on the normal load. Scanning electron microscopy studies revealed that this transition was marked by cracking and disruption of the surface oxide film. A model was proposed that coupled the oxidation rate kinetics of titanium at 650 C with the occurrence of wear at the surface of the oxide film.

Bill, R. C.↗

NASA NTRS1978

Fretting wear of iron, nickel, and titanium under varied environmental conditions

Fretting wear experiments were conducted on high purity iron, nickel and titanium in air under conditions of varied humidity and temperature, and in nitrogen. For iron and titanium, maximum fretting occurred at 10 and 30 percent relative humidity respectively. Nickel showed a minimum in fretting wear at about 10 percent relative humidity. With increasing temperature, all three metals initially showed reduced fretting wear, with increasing wear observed as temperatures increased beyond 200-300 C. For titanium, dramatically reduced fretting wear was observed at temperatures above 500 C, relatable to a change in oxidation kinetics. All three metals showed much less fretting wear in N2 with the presence of moisture in N2 having a proportionally stronger effect than in air.

Bill, R. C.↗

NASA NTRS1979

Fretting wear of iron, nickel, and titanium under varied environmental conditions

Fretting wear experiments were conducted on high-purity iron, nickel and titanium in air under conditions of varied humidity and temperature, and in nitrogen. For iron and titanium, maximum fretting occurred at 10 and 30 percent relative humidity respectively. Nickel showed a minimum in fretting wear at about 10% relative humidity. With increasing temperature, all three metals initially showed reduced fretting wear, with increasing wear observed as temperatures increased beyond 200-300 C. For titanium, dramatically reduced fretting wear was observed at temperatures above 500 C, relatable to a change in oxidation kinetics. All three metals showed much less fretting wear in N2 with the presence of moisture in N2 having a proportionally stronger effect than in air.

Bill, R. C.↗

NASA NTRS1980

The role of oxidation in the fretting wear process

Fretting experiments were conducted on titanium, a series of Ni-Cr-Al alloys and on some high temperature turbine alloys at room temperature and at elevated temperatures in air and in various inert environments. It was found that, depending on temperature and environment, the fretting behavior of the materials examined could be classified according to four general types of behavior. Briefly, these types of behavior were: (1) the complete absence of oxidation, as in inert environments, generally leading to low rates of fretting wear but high fretting friction; (2) gradual attrition of surface oxide with each fretting stroke, found in these experiments to operate in concert with other dominating mechanisms; (3) rapid oxidation at surface fatigue damage sites, resulting in undermining and rapid disintegration of the load bearing surface; and (4) the formation of coherent, protective oxide film, resulting in low rates of fretting wear. An analytical model predicting conditions favorable to the fourth type of behavior was outlined.

Bill, R. C.↗

NASA NTRS2005

High-Frequency, High-Temperature Fretting Experiments

Fretting is a structural damage mechanism observed when two nominally clamped surfaces are subjected to an oscillatory loading. A critical location for fretting induced damage has been identified at the blade/disk and blade/damper interfaces of gas turbine engine turbomachinery and space propulsion components. The high-temperature, high-frequency loading environment seen by these components lead to severe stress gradients at the edge-of-contact. These contact stresses drive crack nucleation and propagation in fretting and are very sensitive to the geometry of the contacting bodies, the contact loads, materials, temperature, and contact surface tribology (friction). To diagnose the threat that small and relatively undetectable fretting cracks pose to damage tolerance and structural integrity of in-service components, the objective of this work is to develop a well-characterized experimental fretting rig capable of investigating fretting behavior of advanced aerospace alloys subjected to load and temperature conditions representative of such turbomachinery components.

Matlik, J. F.↗

DOE OSTI2019

Effect of Al 2 O 3 coating on fretting wear performance of Zr alloy

Fretting wear caused by flow-induced vibration (FIV) is one of the predominant tribological failure modes in nuclear power plants and industry. This paper presents an experimental study of the effect on fretting performance of alumina (Al 2 O 3 ) coatings produced by atomic layer deposition (ALD) on Zirlo (a Zr alloy), a common nuclear fuel cladding alloy. A coating with uniform thickness of about 750 nm was deposited onto a Zirlo flat. Because the typical thickness of fuel cladding is 1 mm, an ultra-thin coating is required. The hardness and elastic modulus of the ALD coating were measured by nano-indentation techniques. With a pin-on-flat contact configuration in low-amplitude reciprocating contact, fretting performance was measured for uncoated Zirlo, Al2O 3 -coated Zirlo, and Al 2 O 3 -coated Zirlo followed by heat treatment. Results showed that the as-deposited Al 2 O 3 coating provided slight improvement in fretting wear performance. However, the heat-treated coating showed two orders of magnitude reduction in fretting wear of the Zr alloy. Wear in the uncoated material involves some local plastic deformation and debris accumulation at the contact interface. In the Al 2 O 3 -coated material, wear occurred by transfer of transparent Al 2 O 3 oxide into the steel pin counterface. Localized removal of coating from the Zirlo alloy flat resulted in a “shark whale” pattern in a scanning electron microscopy image. There was minimal debris accumulation with the heat-treated sample.

36 MATERIALS SCIENCE↗

DOE OSTI2012

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification that enables localization of recombinant proteins in the biosilica cell wall. Our objective was to functionalize diatom biosilica with a reagent-less biosensor with FRET-based imaging capabilities for signaling. The design of the fusion protein conferring these properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the recombinant chimeric protein was first confirmed in E. coli-expressed proteins, prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of CRY showed 95% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica targeting exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 {mu}M and 142.8 {mu}M, respectively. The addition of the silaffin tag for biosilica localization did not influence the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated biosilica in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand binding and conformational change for FRET-signal generation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

FRET Tutorial

In this tutorial, we present the FRET tool for writing, understanding, formalizing and analyzing requirements. In practice, requirements are typically written in natural language, which is ambiguous and consequently not amenable to formal analysis. Since formal, mathematical notations are unintuitive, requirements in FRET are entered in a restricted, natural language, called FRETish with precise unambiguous meaning. This tutorial explains how requirements can be captured in FRETish and subsequently formalized in temporal logics and in the synchronous data flow language Lustre. We show, through multiple examples, how FRET assists users in understanding FRETish requirements and clarifying subtle semantic issues through English and diagrammatic explanations as well as interactive simulation. FInally, this tutorial describes how FRET can be used to perform realizability checking for identifying conflicting requirements and the connection of FRET with (1) the CoCoSim automated analysis tool for the verification of Simulink and Stateflow models, and (2) the Copilot runtime monitoring tool for the analysis of C programs.

FRET↗

DOE OSTI2019

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. Here, we use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (R g ) and end-to-end distance (R ee ) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the R g and R ee are “uncoupled” (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1974

Fretting of Nickel-Chromium-Aluminum Alloys at Temperatures to 816 C

A series of four nickel-based alloys containing 10 percent and 20 percent chromium in combination with 2 percent and 5 percent aluminum were fretted in dry air at temperatures to 816 C. At all temperatures, the alloys showed far less fretting wear than did high-purity nickel. This was attributed to the formation of protective oxide films on the alloys, the result of the selective oxidation of the alloy constituents. Increasing the aluminum concentration reduced fretting wear at all temperatures. Increasing the chromium concentration from 10 percent to 20 percent resulted in decreased fretting wear at 23 and 540 C, but increased fretting wear at 650 and 816 C.

Bill, R. C.↗

NASA NTRS1984

Ice and debris in the fretted terrain, Mars

Viking moderate and high resolution images along the northern highland margin have been monoscopically and stereoscopically examined in order to study the development of fretted terrain. Young debris aprons around mesas and debris in tributary channels create typical fretted morphologies identical to ancient fretted morphologies. This suggests that the debris-apron process operating relatively recently also shaped the fretted terrain of the past. The debris aprons were lubricated by interstitial ice derived from ground ice. Abundant collapse features suggest that ground ice existed and may have flowed in places. The fretting process has been active for a long period and may be active today. The location of debris aprons in two latitudinal belts may be controlled by atmospheric conditions that permit ice in the region to remain in the ground below depths of about one meter and temperatures warm enough for ice to flow.

Lucchitta, B. K.↗

NASA NTRS1990

Evaluation of RSRM case hardware fretting concerns

Fretting corrosion was first noted on Shuttle flight STS-26. This flight was the first usage of the Redesigned Solid Rocket Motor (RSRM). The occurrence of fretting has since been observed on both the field and factory joints of the RSRM. Fretting is a form of corrosion that occurs at the interface between contacting, highly loaded, metal surfaces when exposed to slight relative vibratory motions. The engineering effort performed to evaluate the effect of fretting on the RSRM case hardware is summarized. Based on the results of this evaluation, several conclusions were made concerning flight safety. Also, recommendations were made concerning trending the effects of multiple generations of fretting damage.

Swauger, Thomas R.↗

NASA NTRS2002

Preliminary Study on Fatigue Strengths of Fretted Ti-48Al-2Cr-2Nb

The fatigue behavior (stress-life curve) of gamma titanium aluminide (Ti-48Al-2Cr-2Nb, atomic percent) was examined by conducting two tests: first, a fretting wear test with a fatigue specimen in contact with a typical nickel-based superalloy contact pad in air at temperatures of 296 and 823 K and second, a high-cycle fatigue test of the prefretted Ti-48Al-2Cr-2Nb fatigue specimen at 923 K. Reference high-cycle fatigue tests were also conducted with unfretted Ti-48Al-2Cr-2Nb specimens at 923 K. All Ti-48Al-2Cr-2Nb fatigue specimens were machined from cast slabs. The results indicate that the stress-life results for the fretted Ti-48Al-2Cr-2Nb specimens exhibited a behavior similar to those of the unfretted Ti-48Al-2Cr-2Nb specimens. The values of maximum stress and life for the fretted specimens were almost the same as those for the unfretted specimens. The resultant stress-life curve for the unfretted fatigue specimens was very flat. The flat appearance in the stress-life curve of the unfretted specimens is attributed to the presence of a high density of casting pores. The fatigue strengths of both the fretted and unfretted specimens can be significantly affected by the presence of this porosity, which can decrease the fatigue life of Ti-48Al-2Cr-2Nb. The presence of the porosity made discerning the effect of fretting damage on fatigue strength and life of the specimens difficult.

Miyoshi, Kazuhisa↗

NASA NTRS2013

FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is called a "lights on" FRET response. The vitamin D aptamer beacon gives a "lights off" or inversely proportional fluorescence response to the amount of vitamin D present in diluted serum. These FRET-aptamer assays are rapid (<30 minutes), sensitive (low ng/mL detection limits), and quite easy to carry out (add sample, mix, and detect in the handheld reader). Benefits include the speed of the assays as well as the small amount of space taken up by the handheld reader and cuvette assays. The aptamer DNA sequences represent novel additional features of the existing (patent-pending) FRET-aptamer assay platform.

Bruno, John G.↗

NASA NTRS2023

From Natural Language Requirements to the Verification of Programmable Logic Controllers: Integrating FRET into PLCverif

PLCverif is an actively developed project at CERN, enabling the formal verification of Programmable Logic Controller (PLC) programs in critical systems. In this paper, we present our work on improving the formal requirements specification experience in PLCverif through the use of natural language. To this end, we integrate NASA’s FRET, a formal requirement elicitation and authoring tool, into PLCverif. FRET is used to specify formal requirements in structured natural language, which automatically translates into temporal logic formulae. FRET’s output is then directly used by PLCverif for verification purposes. We discuss practical challenges that PLCverif users face when authoring requirements and the FRET features that help alleviate these problems. We present the new requirement formalization workflow and report our experience using it on two critical CERN case studies.

FRET↗

NASA NTRS1950

Preliminary Investigation of Fretting

An experimental investigation, using microscopic observation and color motion photomicrographs of the action, was conducted to determine the cause of fretting. Glass and other noncorrosive materials, as well as metals, were used as specimens. A very simple apparatus vibrated convex surfaces in contact with stationary flat surfaces at frequencies of 120 cycles or less than one cycle per second, an amplitude of 0.001 inch, and a load of 0.2 pound. The observations and analysis led to the conclusions that fretting was initiated by the loosening, due to the inherent adhesive forces, of finely divided and apparently virgin material, and that its initiation is independent of vibratory motion or high sliding speeds. The fretting of platinum, glass, quartz, ruby, and mica relegated the role of oxidation as a cause to that of a secondary factor. Fretting occurred to clean nonmetals and metals readily, and glass microscope slides and steel balls provide excellent method for visual studies. Video run time 9:54, color and sound (mp4). Available for download for best viewing. Document indicated in video is related in the repository and can also be downloaded for use at https://ntrs.nasa.gov/citations/19930083206.

fretting↗

NASA NTRSDate not supplied

From Natural Language Requirements to the Verification of Programmable Logic Controllers: Integrating FRET into PLCverif

FRET↗