Trehalose Film Research Articles

Ready-to-use thermally stable mastermix pills for molecular biology applications.

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.

恒温・恒湿環境下におけるアモルファス・トレハロース乾燥膜の 相変化と吸湿率の測定

恒温・恒湿環境下におけるアモルファス・トレハロース乾燥膜の 相変化と吸湿率の測定

Reducing the Matrix Effect in Organic Cluster SIMS Using Dynamic Reactive Ionization.

Dynamic reactive ionization (DRI) utilizes a reactive molecule, HCl, which is doped into an Ar cluster projectile and activated to produce protons at the bombardment site on the cold sample surface with the presence of water. The methodology has been shown to enhance the ionization of protonated molecular ions and to reduce salt suppression in complex biomatrices. In this study, we further examine the possibility of obtaining improved quantitation with DRI during depth profiling of thin films. Using a trehalose film as a model system, we are able to define optimal DRI conditions for depth profiling. Next, the strategy is applied to a multilayer system consisting of the polymer antioxidants Irganox 1098 and 1010. These binary mixtures have demonstrated large matrix effects, making quantitative SIMS measurement not feasible. Systematic comparisons of depth profiling of this multilayer film between directly using GCIB, and under DRI conditions, show that the latter enhances protonated ions for both components by 4- to ~15-fold, resulting in uniform depth profiling in positive ion mode and almost no matrix effect in negative ion mode. The methodology offers a new strategy to tackle the matrix effect and should lead to improved quantitative measurement using SIMS. Graphical Abstract ᅟ.

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

Long-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells.

A mixed cluster ion beam to enhance the ionization efficiency in molecular secondary ion mass spectrometry

Chemical modification of a rare gas cluster ion beam (GCIB) to increase the intensity of desorbed molecular ions in secondary ion mass spectrometry experiments relative to the pure Ar cluster. Doping of the GCIB by mixing small concentration levels (1-3% relative partial pressure) of CH4 into the Ar gas driving the cluster ion source. Mass spectra measured on a trehalose film using the doped GCIB exhibit enhanced molecular ion signals. From depth profiling experiments, the results are shown to arise from an increase in the ionization efficiency of the sputtered molecules rather than a change in the sputtering yield of neutral species. Tuning of the chemistry of mixed clusters is suggested as a general approach to enhancing the ionization probability of sputtered molecules.

Investigations of molecular depth profiling with dual beam sputtering

ABSTRACTIn this study, the feasibility of molecular depth profiling using dual beam sputtering of biological materials is examined. The model system is a 402‐nm Langmuir Blodgett multilayer film consisting of 149 monolayers of barium arachidate (AA). The thin film was initially subjected to pre‐bombardment with a 15‐keV Au+ beam. Subsequently, an imaging depth profile experiment was performed on the pre‐irradiated sample using a 40‐keV C60+ beam. An extremely low erosion rate under Au+ bombardment is found on this model system. In the subsequent C60+ depth profiles, surprisingly large molecular ion signals are detected at the gold pre‐irradiated surface. These signals then rapidly decay to nearly zero, indicating a damaged sub‐surface layer being generated by the Au+ pre‐bombardment. The thickness of the damaged layer is found to increase with increasing gold ion fluence and saturate at about 100 nm at 6 × 1014 Au+/cm2. This altered layer thickness is significantly larger than the value of ~50 nm obtained on a trehalose film pre‐bombarded with a Ga+ source. The results also show that the damage caused by the Au+ beam can be removed by C60+ sputtering and that the molecular information is restored after damage removal. Copyright © 2012 John Wiley & Sons, Ltd.

Exploring the surface sensitivity of TOF-secondary ion mass spectrometry by measuring the implantation and sampling depths of Bi(n) and C60 ions in organic films.

The surface sensitivity of Bi(n)(q+) (n = 1, 3, 5, q = 1, 2) and C(60)(q+) (q = 1, 2) primary ions in static time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments were investigated for molecular trehalose and polymeric tetraglyme organic films. Parameters related to surface sensitivity (impact crater depth, implantation depth, and molecular escape depths) were measured. Under static TOF-SIMS conditions (primary ion doses of 1 × 10(12) ions/cm(2)), the 25 keV Bi(1)(+) primary ions were the most surface sensitive with a molecular escape depth of 1.8 nm for protein films with tetraglyme overlayers, but they had the deepest implantation depth (~18 and 26 nm in trehalose and tetraglyme films, respectively). The 20 keV C(60)(+2) primary ions were the second most surface sensitive with a slightly larger molecular escape depth of 2.3 nm. The most important factor that determined the surface sensitivity of the primary ion was its impact crater depth or the amount of surface erosion. The most surface sensitive primary ions, Bi(1)(+) and C(60)(+2), created impact craters with depths of 0.3 and 1.0 nm, respectively, in tetraglyme films. In contrast, Bi(5)(+2) primary ions created impact craters with a depth of 1.8 nm in tetraglyme films and were the least surface sensitive with a molecular escape depth of 4.7 nm.

Ionization effects in molecular depth profiling of trehalose films using buckminsterfullerene (C60) cluster ions.

Salts play a mysterious role in desorption mass spectrometry, especially in biological samples.[1] We used trehalose films doped with a peptide as a well defined model system to investigate the ionization effects in organic molecular depth profiling. Sodium salts at 1% level were added into the solution used to produce the trehalose films, and depth profiles were obtained with a C60 ion source. The results show that the protonated molecular ion signal from the peptide and the quasimolecular ion signal of trehalose are significantly suppressed by the addition of salts, whereas the signals representing salt clusters and salt adducts of trehalose are formed in both positive and negative modes. The formation of protonated molecular ions is found to correlate with the ratio between protonated and bare water ions, suggesting that the latter can be used as an indicator for the accumulation of protons liberated by the ion bombardment. In experiments where no salt was added, it is shown that the surface variation of the protonated molecular ion signal strongly depends upon the water content of the trehalose film.

ToF‐SIMS depth profiling of trehalose: the effect of analysis beam dose on the quality of depth profiles

AbstractIn static SIMS experiments, an analysis dose of 1012 ions/cm2 typically produces optimum results. However, the same dose used in dual beam depth profiling can significantly degrade the signal. This is because during each analysis cycle a high‐energy beam is rastered across the same x‐y location on the sample. If a sufficient amount of sample is not removed during each sputter cycle, the subsequent analysis cycle will sample a volume degraded by the previous analysis cycles. The dimensionless parameter, R′, is used to relate the amount of damage accumulated in the sample to the amount of analysis beam dose used relative to the etching beam. Depth profiles from trehalose films spin‐cast onto silicon wafers acquired using Bi1+ and Bi3+ analysis beams were compared. As R′ increased, the depth profile and the depth resolution (interface width) both degraded. At R′ values below 0.04 for both Bi1+ and Bi3+, the shape of the profile as well as the depth resolution (9 nm) indicated that dual beam analysis can be superior to C60 single beam depth profiling. Copyright © 2010 John Wiley & Sons, Ltd.

Preparation and characterisation of dry thin native protein trehalose films on titanium-coated cyclo-olefin polymer (COP) foil generated by spin-coating/drying process and applied for protein transfer by Laser-Induced-Forward Transfer (LIFT)

Laser-Induced-Forward Transfer (LIFT) is a non-contact method for transferring bioactive substances such as proteins from a so-called LIFT target, here a titanium-coated cyclo-olefin polymer (COP) foil, to a surface of a solid work piece, e.g. a medical implant. The quality of the surface coating of the LIFT target, i.e. its homogeneity and the bioactivity of the ingredients to be transferred, is crucial for the quality and the efficiency of the overall process. Here, aqueous trehalose solutions containing the proteins streptavidin, green fluorescent protein (GFP), and bovine serum albumin were used in a spin-coating/drying process to render dry thin native protein trehalose films on titanium-coated COP foils. The homogeneity of the layer thicknesses was characterised by spectroscopic ellipsometry and the homogeneity of the GFP distribution within the layer was monitored by fluorescence scan analyses. The bioactivity of the embedded proteins was probed after short- and long-term storage by FT-IR spectroscopy in spectral reflection method and biological activity tests based on fluorescence scan analyses. After application of the LIFT process, the transferred proteins were tested in their bioactivity by fluorescence scan analyses and their topology by AFM analysis, proving that highly functional LIFT targets can be prepared by the chosen method.

Three-dimensional depth profiling of molecular structures

Molecular time of flight secondary ion mass spectrometry (ToF-SIMS) imaging and cluster ion beam erosion are combined to perform a three-dimensional chemical analysis of molecular films. The resulting dataset allows a number of artifacts inherent in sputter depth profiling to be assessed. These artifacts arise from lateral inhomogeneities of either the erosion rate or the sample itself. Using a test structure based on a trehalose film deposited on Si, we demonstrate that the "local" depth resolution may approach values which are close to the physical limit introduced by the information depth of the (static) ToF-SIMS method itself.

Molecular Depth Profiling using a C(60) Cluster Beam: the Role of Impact Energy.

Molecular depth profiling of organic overlayers was performed using a mass selected C(60) ion beam in conjunction with time-of-flight (TOF-SIMS) mass spectrometry. The characteristics of sputter depth profiles acquired for a 300-nm Trehalose film on silicon were studied as a function of the kinetic impact energy of the projectile ions. The results are interpreted in terms of a simple model describing the balance between sputter erosion and ion induced chemical damage. It is shown that the efficiency of the projectile to clean up the fragmentation debris produced by its own impact represents a key parameter governing the success of molecular depth profile analysis.

Molecular depth profiling of trehalose using a C 60 cluster ion beam

Molecular depth profiling of organic overlayers was performed using a mass selected fullerene ion beam in conjunction with time-of-flight (TOF-SIMS) mass spectrometry. The characteristics of depth profiles acquired on a 300-nm trehalose film on Si were studied as a function of the impact kinetic energy and charge state of the C 60 projectile ions. We find that the achieved depth resolution depends only weakly upon energy.

Three-dimensional molecular imaging using mass spectrometry and atomic force microscopy

We combine imaging ToF-SIMS depth profiling and wide area atomic force microscopy to analyze a test structure consisting of a 300 nm trehalose film deposited on a Si substrate and pre-structured by means of a focused 15-keV Ga + ion beam. Depth profiling is performed using a 40-keV C 60 + cluster ion beam for erosion and mass spectral data acquisition. A generic protocol for depth axis calibration is described which takes into account both lateral and in-depth variations of the erosion rate. By extrapolation towards zero analyzed lateral area, an “intrinsic” depth resolution of about 8 nm is found which appears to be characteristic of the cluster-surface interaction process.

Protocols for Three-Dimensional Molecular Imaging Using Mass Spectrometry

A protocol for three-dimensional molecular thin-film analysis is described that utilizes imaging time-of-flight secondary ion mass spectrometry and large-area atomic force microscopy. As a test study, a 300-nm trehalose film deposited on a Si substrate was structured by bombardment with a focused 15-keV Ga+ ion beam and analyzed using a 40-keV C60+ cluster ion beam. A three-dimensional sputter depth profile was acquired as a series of high-resolution lateral SIMS images with intermittent erosion cycles. As the most important result of this study, we find that the structured film exhibits a highly nonuniform erosion rate, thus preventing a simple conversion of primary ion fluence into eroded depth. Instead, the depth scale calibration must be performed individually on each pixel of the imaged area. The resulting laterally resolved depth profiles are discussed in terms of the chemical damage induced by the Ga+ bombardment along with the physics of the C60+ induced erosion process.

Depth Profiling of Peptide Films with TOF-SIMS and a C60 Probe

A buckminsterfullerene ion source is employed to characterize peptide-doped trehalose thin films. The experiments are designed to utilize the unique sputtering properties of cluster ion beams for molecular depth profiling. The results show that trehalose films with high uniformity can be prepared on Si by a spin-coating technique. Bombardment of the film with C60+ results in high quality time-of-flight secondary ion mass spectrometry spectra, even during ion doses of up to 3 x 10(14) ions/cm2. This result is in contrast to atomic bombardment experiments in which the dose of incident ions must be kept below 10(12) ions/cm2 so as to retain mass spectral information. Moreover, since the films are of uniform thickness, it is possible to depth-profile through the film and into the Si substrate. This experimental protocol allows the yield of trehalose molecular equivalents and the degree of interface mixing to be evaluated in detail. When doped with a variety of small peptides up to a molecular weight of m/z 500, we find that the peptide molecular ion intensity remains stable under continuous C60+ bombardment, although some decrease in intensity is observed. The results are interpreted in terms of a model whereby the high trehalose yield and low damage depth of the C60 projectile combine to prevent damage accumulation. In general, the peptide-trehalose system provides a valuable model for evaluating the parameters that lead to effective 3-dimensional characterization of biomaterials.

Protein in Sugar Films and in Glycerol/Water as Examined by Infrared Spectroscopy and by the Fluorescence and Phosphorescence of Tryptophan

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20–300K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at ∼160K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20–300K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.

Solvent effects on conformational dynamics of proteins: Cytochrome c in a dried trehalose film

The spectral diffusion dynamics of free base cytochrome c (H2-Cc) in a dry trehalose film is tremendously enhanced as compared to a glycerol/water glass. We show that relaxation as well as fluctuation processes contribute to the spectral diffusion dynamics. Relaxation shows up in aging phenomena which can be measured in a separate fashion. In both solvents, the spectral diffusion as well as the aging dynamics follow power laws in time. The respective exponents are quite different, yet the influence of the solvent on them is only marginal. The large difference in the magnitude of the spectral diffusion dynamics in the two solvents can be traced back to a correspondingly large difference in the inhomogeneous width which itself seems to come from the much higher glass transition temperature in trehalose, which is close to the unfolding temperature of cytochrome c.

Stabilization of dry immobilized acetylcholinesterase on microtitration plates for colorimetric determination of its inhibitors in water and biological fluids

The ability of the gelatin (or BSA)-trehalose film to convert normally fragile dry immobilized acetylcholinesterase enzyme (AchE) into a stable reagent is shown. The remarkable properties of the dry immobilized AchE enzyme preparation are its stability to prolonged exposure to temperature as high as +50°C and its tolerance to damages due to acidic pH. The proposed method offers a rapid, simple, and inexpensive means for mass screening of AchE inhibitor residues such as pesticides and drugs in water, vegetables, and human blood serum.