Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy

Raman spectroscopy was tested for the identification of biomolecules (glycine, L-alanine, β-alanine, L-serine, and γ-aminobutyric acid) trapped in fluid inclusions inside halite model crystals. The investigated biomolecules represent important targets for future astrobiological missions. We know from terrestrial conditions that organic molecules and microorganisms can be sealed within fluid inclusions and can survive intact even for hundreds of millions of years. Raman spectroscopy is currently being miniaturized for future extraterrestrial planetary exploration (ExoMars 2018). Raman spectroscopy has shown the ability to detect investigated aminoacids nondestructively without any sample preparation, in short measurement times, and in relatively low concentrations. The number of registered Raman bands of investigated aminoacids and their intensity clearly correlate with the given concentration of biomolecules within fluid inclusions.     

Combined time- and space-resolved Raman spectrometer for the non-invasive depth profiling of chemical hazards

A time-resolved inverse spatially offset Raman spectrometer was constructed for depth profiling of Raman-active substances under both the lab and the field environments. The system operating principles and performance are discussed along with its advantages relative to traditional continuous wave spatially offset Raman spectrometer. The developed spectrometer uses a combination of space- and time-resolved detection in order to obtain high-quality Raman spectra from substances hidden behind coloured opaque surface layers, such as plastic and garments, with a single measurement. The time-gated spatially offset Raman spectrometer was successfully used to detect concealed explosives and drug precursors under incandescent and fluorescent background light as well as under daylight. The average screening time was 50 s per measurement. The excitation energy requirements were relatively low (20 mW) which makes the probe safe for screening hazardous substances. The unit has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than previous picosecond-based systems, to provide a functional platform for in-line or in-field sensing of chemical substances.     

Intracavity laser polarization spectrometer for biomolecule traces

A polarization variant of an intracavity laser spectrometer based on the competition of beams in an argon ion laser, radiating a number of narrow lines in the 275 to 529 nm spectral range, is proposed and described for the spectral analysis of liquid samples, having broad absorption bands, mainly biomolecules in solutions. By careful optimization of the operating conditions, a procedure has been established that results in a minimal measurable absorbance decreased up to 10^–5 units of optical density. The examined spectrometer provides a wide dynamic range of analysis. A determination of trace contents of amino acids in methanol were carried out.     

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L⁻¹. The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86 ± 4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78 ± 8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L⁻¹. Comparison of the fermentation efficiencies measured by Raman spectroscopy (80 ± 10%) and gas chromatrography-mass spectrometry (87 ± 9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.     

Chemiluminescence from singlet oxygen that was detected at two wavelengths and effects of biomolecules on it

We developed the detection apparatus that equipped with the two-photomultiplier tubes for chemiluminescence from singlet oxygen. Singlet oxygen was generated with reaction between sodium hypochlorite and hydrogen peroxide. The chemiluminescence from singlet oxygen, the dimol light emission (ca. 634 nm) and the monomol light emission (ca. 1270 nm), was observed simultaneously for the same reaction cell. The effects of sodium azide as an antioxidant, human serum albumin, and α-amino acids on the chemiluminescence based on the both emissions were examined; the observed chemiluminescence could provide direct information with regard to the reaction between singlet oxygen and antioxidant/biomolecules. The apparent rate constants for quenching singlet oxygen in the presence of human serum albumin were calculated to be ca. 3.3 × 10⁹ and ca. 8.8 × 10⁸ M⁻¹ s⁻¹ for the dimol and monomol light emissions, respectively, under the present conditions. The chemiluminescence intensities of the dimol emission decreased in the presence of His, Asp, Phe, Ser, and Tyr, and that of the monomol decreased in the presence of Cys and Trp. The chemiluminescence observed in the presence of biomolcules was discussed together with the reactivities of sodium hypochlorite and hydrogen peroxide to biomolecules.     

Comparison of the Possibilities of Thermal-Lens Detection in Capillaries and Microchips

The conditions of thermal-lens detection are compared for samples with small path lengths: capillaries and microchannels of chemical microchips. The optimum parameters of the optical configuration of a thermal lens spectrometer (beam diameters and their in-sample ratios, and the detector position) were calculated from the theory and confirmed experimentally. The limits of detection of a model chemical substance (ferroin) are estimated at 1 × 10^–8 M for microchips and 5 × 10^–8 M for capillaries. The absolute limits of detection are the same and equal to 3 × 10^–17 M. The advantages and disadvantages of thermal lens detection in capillaries and microchips are discussed.     

Surface-Enhanced Raman Spectroscopy (SERS) for characterization SARS-CoV-2

We used SERS with silver nanoparticles (AgNPs) as the active substrate to develop a, simple, quick, and accurate method for the detection and characterization SARS-CoV-2 without the need for RNA isolation and purification. Inactivated SARS-CoV-2 was used. The SERS signals were more than 10⁵ times enhanced than the normal Raman (NR) spectra. The SERS spectra of SARS-CoV-2 fingerprint revealed pronounced intensity signals of nucleic acids; aromatic amino acid side chains: 1007 cm^?1 (Phe marker), 1095 cm^?1 (CN and PO₂^? markers), 1580 cm^?1 (Tyr, Trp markers). Vibrations of the protein main chain: 1144 cm^?1 (CN and NH₂ markers), 1221 cm^?1 (CN and NH markers), 1270 cm^?1 (NH₂ marker), 1453 cm^?1 (CHCH₂ marker). All of these biomolecules could be adsorbed on the AgNPs surface's dense hot patches. The intensity of the SERS band varied with the concentration of SARS-CoV-2, with a virus detection limit of less than 10³ vp/mL and RSDs of 20 %.     

Identification of β-carotene in an evaporitic matrix—evaluation of Raman spectroscopic analysis for astrobiological research on Mars

Since evaporitic rocks on the Martian surface could (or still can) serve as potential habitats for microbial life on Mars, there is a reasonable possibility that these rocks may sustain molecular remnants as evidence for the presence of extinct or extant living organisms on Mars and that β-carotene could be a suitable biomarker. In this paper, Raman microspectrometry was tested as a nondestructive method of determining the lowest detectable β-carotene content in experimentally prepared evaporitic matrices—namely, gypsum, halite and epsomite. Two excitation wavelengths were compared—514.5 nm, because of the resonance Raman enhancement in the carotenoid analysis, and 785 nm, as a more universal wavelength now much used in the detection of biomolecules terrestrially. Mixtures were measured directly as well as with a laser beam penetrating the crystals of gypsum and epsomite. We have obtained β-carotene signals at the 0.1 to 10 mg kg⁻¹ level—the number of registered β-carotene Raman bands differed depending on the particular mineral matrix and the excitation wavelength. Concentrations of β-carotene of about one order of magnitude higher were identified when analysed through single crystals of gypsum and epsomite, respectively.     

Activation of Nanoparticles by Biosorption for <Emphasis Type="Italic">E. coli</Emphasis> Detection in Milk and Apple Juice

Two types of silver nanoparticles were activated by specific sorption of biomolecules for the detection of Escherichia coli. The capture of this bacterium was performed using polyclonal antibodies (anti-E. coli) biosorbed onto nanospheres or nanorice through a protein-A layer. The bacterial detection was achieved using surface enhancement Raman scattering in order to compare the performance of these two nanoparticles. The activated silver nanospheres showed a better performance mainly due to the dimension of these nanoparticles. The detection limit has been established using the automated Raman mapping system. The technique was capable of detecting 10³ cells/mL in milk and apple juice without any pre-enrichment. With an overall assay time less than 1 h, the process could be easily adapted to detect other pathogens by selecting the pertinent antibody. Furthermore, PCR was used for the DNA verification to assess whether the selected bacterial strain was identical before and after detection.     

Critical evaluation of a handheld Raman spectrometer with near infrared (785nm) excitation for field identification of minerals

Handheld Raman spectrometers (Ahura First Defender XL, Inspector Raman DeltaNu) permit the recording of acceptable and good quality spectra of a large majority of minerals outdoors and on outcrops. Raman spectra of minerals in the current study were obtained using instruments equipped with 785 nm diode lasers. Repetitive measurements carried out under an identical instrumental setup confirmed the reliability of the tested Raman spectrometers. Raman bands are found at correct wavenumber positions within ±3 cm(-1) compared to reference values in the literature. Taking into account several limitations such as the spatial resolution and problems with metallic and black and green minerals handheld Raman spectrometers equipped with 785 nm diode lasers can be applied successfully for the detection of minerals from the majority of classes of the mineralogical system. For the detection of biomarkers and biomolecules using Raman spectroscopy, e.g. for exobiological applications, the near infrared excitation can be considered as a preferred excitation. Areas of potential applications of the actual instruments include all kind of common geoscience work outdoors. Modified Raman systems can be proposed for studies of superficial or subsurface targets for Mars or Lunar investigations.     

In situ detection of a single carotenoid crystal in a plant cell using Raman microspectroscopy

It is the first report of direct, in situ detection of carotenoids at the subcellular level by using Raman microspectroscopy. Single crystals sequestered in a carrot cell were measured using FT-Raman spectrometer equipped with a microscope and 40× objective. The observed characteristic bands centered at 1518 cm⁻¹ and 1156 cm⁻¹ proved the crystals were composed of carotenoids with β-carotene being predominant. The obtained results show the potential of Raman microspectroscopy for identification and analysis of compounds localized in cytoplasm by taking measurements directly from a single plant cell.     

Raman sensitivity enhancement for aqueous absorbing sample using Teflon-AF 2400 liquid core optical fibre cell

The compatibility Teflon-AF 2400 liquid core optical fibre with resonance Raman spectroscopy (RRS-LCOF) was used to detect aqueous biomolecules. The maximum sensitivity enhancement factor for concentrations greater than the detection limit in a conventional cell was 10, and detection limit reduction of about 1000-fold have been achieved for the measurement of aqueous absorbing sample using Teflon-AF 2400 fibre Raman cell compared to the conventional cell. We were able to collect spectra of 2.5 × 10⁻⁹ and 2.5 × 10⁻¹⁰ M aqueous β-carotene using 16.2 mW of laser power and 10 s integration time. This volume of a 2.5 × 10⁻¹⁰ M aqueous solution corresponds to only 1.5 fmol or 830 fg of β-carotene. The results of this preliminary study indicate that RRS-LCOF has potential in bioanalytical and biomedical applications.     

Chemiluminescence-based detection technologies for biomolecules, mainly in gel electrophoresis

We provide an overview of powerful applications of chemiluminescence (CL) analysis for biomolecules, mainly in gel electrophoresis. In routine immunoassays, CL labels and detection reactions are widely used for peroxidase and alkaline-phosphatase enzymes. The sensitivity, the dynamic range and the diversity of CL assays have led to a vast range of applications, notably in protein and nucleic-acid blotting. Non-enzymatic CL detection is also being developed gradually.Direct CL detection of biomolecules in gels has emerged recently, with simple, convenient and rapid methods. It offers substantial potential to detect many proteins or nucleic acids in complex biological samples. In addition, metallic nanoparticles and catalytic nucleic acids are also potential candidates for CL detection of biomolecules in the future.     

Estimation of gas‐phase acidities of a series of dicarboxylic acids by the kinetic method

The gas‐phase acidities (GA) of a series of dicarboxylic acids were estimated by applying the extended kinetic method. Proton‐bound heterodimeric anions [A^?H⁺B^?] of a series of dicarboxylic acids (A) and reference compounds (B) were generated under electrospray ionization conditions, and the dissociation of these cluster ions was examined using a triple‐quadrupole mass spectrometer. The mass‐selected proton‐bound heterodimeric anions were fragmented to yield individual monomer anions as the only product ions, and the abundance ratios of these anions were used to estimate the GA values of the dicarboxylic acids. The experiment was performed over a range of collision energies in order to more accurately determine GA values and to estimate the differences in the entropy of deprotonation of the dicarboxylic acids and the reference compounds. The trends in GA values obtained for the dicarboxylic acids could imply cyclization of the structures via intramolecular hydrogen bonding. The lower homologues show higher GA values than the higher homologues. The GA order for lower homologues is comparable with that of their solution‐phase pKa1 values. Copyright © 2005 John Wiley & Sons, Ltd.     

Fourier transform Raman spectroscopy in the visible region

Low-resolution (ca. 4cm^–1) Raman spectra of various liquids and solids, excited by an argon laser at 448 and 514.5 nm, were recorded using a BOMEM DA3.02 commercial interferometer. These spectra were compared with those obtained in the same experimental conditions on a conventional dispersive spectrometer. It is concluded that for low-resolution Raman studies with excitation in the visible region, the dispersive method is superior to the interferometric technique.     

Raman spectroscopic study of bacterial endospores

Endospores and endospore-forming bacteria were studied by Raman spectroscopy. Raman spectra were recorded from Bacillus licheniformis LMG 7634 at different steps during growth and spore formation, and from spore suspensions obtained from diverse Bacillus and Paenibacillus strains cultured in different conditions (growth media, temperature, peroxide treatment). Raman bands of calcium dipicolinate and amino acids such as phenylalanine and tyrosine are more intense in the spectra of sporulating bacteria compared with those of bacteria from earlier phases of growth. Raman spectroscopy can thus be used to detect sporulation of cells by a characteristic band at 1,018 cm^–1 from calcium dipicolinate. The increase in amino acids could possibly be explained by the formation of small acid-soluble proteins that saturate the endospore DNA. Large variations in Raman spectra of endospore suspensions of different strains or different culturing conditions were observed. Next to calcium dipicolinate, tyrosine and phenylalanine, band differences at 527 and 638 cm^–1 were observed in the spectra of some of the B. sporothermodurans spore suspensions. These bands were assigned to the incorporation of cysteine residues in spore coat proteins. In conclusion, Raman spectroscopy is a fast technique to provide useful information about several spore components. Figure A difference spectrum between Raman spectra of B. licheniformis LMG 7634 cultured for 6 days and 1 day, together with the reference Raman spectrum of calcium dipicolinate     

Search for evidence of life in space: Analysis of enantiomeric organic molecules by N,N-dimethylformamide dimethylacetal derivative dependant Gas Chromatography–Mass Spectrometry

Within the context of the future space missions to Mars (MSL 2011 and Exomars 2016), which aim at searching for traces of life at the surface, the detection and quantitation of enantiomeric organic molecules is of major importance. In this work, we have developed and optimized a method to derivatize and analyze chiral organic molecules suitable for space experiments, using N,N-dimethylformamide dimethylacetal (DMF-DMA) as the derivatization agent. The temperature, duration of the derivatization reaction, and chromatographic separation parameters have been optimized to meet instrument design constraints imposed upon space experiment devices. This work demonstrates that, in addition to its intrinsic qualities, such as production of light-weight derivatives and a great resistance to drastic operating conditions, DMF-DMA facilitates simple and fast derivatization of organic compounds (three minutes at 140 °C in a single-step) that is suitable for an in situ analysis in space. By using DMF-DMA as the derivatization agent, we have successfully identified 19 of the 20 proteinic amino acids and been able to enantiomerically separate ten of the potential 19 (glycine being non-chiral). Additionally, we have minimized the percentage of racemized amino acid compounds produced by optimizing the conditions of the derivatization reaction itself. Quantitative linearity studies and the determination of the limit of detection show that the proposed method is also suitable for the quantitative determination of both enantiomeric forms of most of the tested amino acids, as limits of detection obtained are lower than the ppb level of organic molecules already detected in Martian meteorites.     

Surface enhanced Raman scattering for multiplexed detection

The multiplexed detection of biological analytes from complex mixtures is of crucial importance for the future of intelligent management and detection of disease. This review focuses on recent advances in the use of surface enhanced Raman scattering (SERS) spectroscopy as an analytical technique that can deliver multiplexed detection for a variety of biological target in increasingly complex media. The use of SERS has developed from the multipelxed detection of custom dye molecules to biomolecules such as DNA and proteins. Recent work has also shown the capability of SERS multiplexing for in vivo as well as in vitro applications.     

1064 nm dispersive multichannel Raman spectroscopy for the analysis of plant lignin

The mixed phenylpropanoid polymer lignin is one of the most abundant biopolymers on the planet and is used in the paper, pulp and biorenewable industries. For many downstream applications, the lignin monomeric composition is required, but traditional methods for performing this analysis do not necessarily represent the lignin composition as it existed in the plant. Herein, it is shown that Raman spectroscopy can be used to measure the lignin monomer composition. The use of 1064 nm excitation is needed for lignin analyses since high fluorescence backgrounds are measured at wavelengths as long as 785 nm. The instrument used for these measurements is a 1064 nm dispersive multichannel Raman spectrometer that is suitable for applications outside of the laboratory, for example in-field or in-line analyses and using remote sensing fiber optics. This spectrometer has the capability of acquiring toluene/acetonitrile spectra with 800 cm⁻¹ spectral coverage, 6.5 cm⁻¹ spectral resolution and 54 S/N ratio in 10 s using 280 mW incident laser powers. The 1135–1350 cm⁻¹ and 1560–1650 cm⁻¹ regions of the lignin spectrum can be used to distinguish among the three primary model lignin monomers: coumaric, ferulic and sinapic acids. Mixtures of the three model monomers and first derivative spectra or partial least squares analysis of the phenyl ring breathing modes around 1600 cm⁻¹ are used to determine sugarcane lignin monomer composition. Lignin extracted from sugarcane is shown to have a predominant dimethoxylated and monomethoxylated phenylpropanoid content with a lesser amount of non-methoxylated phenol, which is consistent with sugarcane's classification as a non-woody angiosperm. The location of the phenyl ring breathing mode peaks do not shift in ethanol, methanol, isopropanol, 1,4 dioxane or acetone.     

Volume exclusion influences in spectral characteristics of DNA-amino acids complexes

Living cells contain a variety of bio molecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These bio molecules occupy a significant fraction (20–40%) of the cellular volume. The total concentration of biomolecules reaches 400 g l⁻¹, leading to a crowded intracellular environment referred to as molecular crowding. This causes changes in chemical potential strongly affecting the physicochemical properties in the cell environment. Hence understanding the effects of molecular crowding conditions on biomolecules and biomolecular complexes is of utmost importance. In the present study, interaction of DNA with various amino acids has been observed under the influence of molecular crowding agents of various molecular sizes to understand the changes in DNA characteristics with reference to binding parameters to various amino acids using fluorescence and FTIR spectra. These studies are expected to indicate the changes in the DNA-amino acid complexes with particular significance of effects of molecular crowding in response to volume exclusion effects.