Potential of Raman spectroscopic techniques to study proteins.

Proteins are large, complex molecules responsible for various biological processes. However, protein misfolding may lead to various life-threatening diseases. Therefore, it is vital to understand the shape and structure of proteins. Despite numerous techniques, a mechanistic understanding of the protein folding process is still unclear. Therefore, new techniques are continually being explored. In the present article, we have discussed the importance of Raman spectroscopy, Raman Optical Activity (ROA) and various other advancements in Raman spectroscopy to understand protein structure and conformational changes based on the review of our earlier work and recent literature. A Raman spectrum of a protein provides unique signatures for various secondary structures like helices, beta-sheets, turns, random structures, etc., and various amino acid residues such as tyrosine, tryptophan, and phenylalanine. We have shown how Raman spectra can differentiate between bovine serum albumin (BSA) and lysozyme protein based on their difference in sequence and structure (primary, secondary and tertiary). Although it is challenging to elucidate the structure of a protein using a Raman spectrum alone, Raman spectra can be used to differentiate small changes in conformations of proteins such as BSA during melting. Various new advancements in technique and data analyses in Raman spectroscopic studies of proteins have been discussed. The last part of the review focuses on the importance of the ROA spectrum to understand additional features about proteins. The ROA spectrum is rich in information about the protein backbone due to its rigidity compared to its side chains. Furthermore, the ROA spectra of lysozyme and BSA have been presented to show how ROA provides extra information about the solvent properties of proteins.

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis.

BACKGROUND AND OBJECTIVES: Despite having numerous advances in therapeutics, mortality and morbidity due to oral cancer incidence are still very high. Early detection can improve the chances of survival in most patients. However, diagnosis at early stages can be challenging as premalignant conditions are usually asymptomatic. Currently, histological assessment remains the gold standard for diagnosis. Early diagnosis poses challenges to pathologists due to less severe morphological changes associated with early stages. Therefore, a fast and robust method of detection based on molecular changes is needed for early diagnosis. © 2021 Wiley Periodicals LLC. STUDY DESIGN/MATERIAL AND METHODS: In the present study, Fourier transform infrared (FTIR) spectroscopic imaging has been used to differentiate early-stage oral hyperplasia from adjacent normal (AN) and oral squamous cell carcinoma (OSCC). Hyperplasia is often considered as an initial event in the pathogenesis of oral cancer and OSCC is the most common advanced stage of malignancy. Differentiating normal versus hyperplasia and hyperplasia versus OSCC can remain quite challenging on occasion using conventional staining as the histological assessment is based on morphological changes. RESULTS: Unsupervised hierarchical cluster analysis (UHCA) has been performed on FTIR images of multiple tissues together that provided some degree of classification among tissue groups. The AN epithelium clustered distinctively using UHCA from both hyperplasia and grades 1 and 2 of OSCC. An increase in the content of DNA, denaturation of protein, and altered lipid structures were more clearly elucidated with spectral analysis. CONCLUSION: This study demonstrates a simple strategy to differentiate early-stage oral hyperplasia from AN and OSCC using UHCA. This study also proposes a future alternative method where FTIR imaging can be used as a diagnostic tool for cancer at early stages.

Understanding phase transition and vibrational mode coupling in ammonium nitrate using 2D correlation Raman spectroscopy.

Ammonium nitrate (AN) is an important component of the chemical industry such as an active ingredient in fertilizers, as an oxidizer in explosive compositions and propellants, and as a blasting agent in civil explosives. Numerous accidents have been reported in the past which concerns its thermal instability and poses a big threat to its processing, transportation, and storage. Despite much literature being reported to understand its thermal instability, a mechanistic view remains unclear. In the present work, we have studied the behavior of AN to temperature change using a mathematical approach called 2D correlation (2D Cos) Raman spectroscopy to provide complete insight into the detailed dynamical nature of the interactions between the species (ionic or molecular) occurring with an increase in temperature. We have analyzed various libration and translational modes of nitrate in the low-frequency region using this mathematical tool. It is observed from 2D maps that the phase transition of AN starts with changes in libration modes followed by various nitrate modes and ammonium modes which further precedes low-frequency translational modes. Further, the 2D correlation could differentiate between modes splitting and shifting based on specific 2D Cos pattern. The changes occurring in the N-O deformation modes, symmetric stretching modes as well as anti-symmetric stretching modes which have been attributed to the weakening of the hetero-ionic coupling between the NH4+ and the NO3- ions could be clearly distinguished in the 2D synchronous and asynchronous plots. Besides, moving window analysis was performed to visualize the transition temperature at which phase change of AN takes place.

Probing the Stepwise Unfolding of Bovine Serum Albumin Using 2D Correlation Raman Spectroscopic Analysis.

Protein denaturation involves a change in the protein structure with the loss of activity, which proceeds via various intermediates. The possible intermediate structures account largely for understanding the process of unfolding. Hence, considerable attention is required to characterize partially unfolded protein states and to gain more insight into the information about the sequence and steps involved in protein folding mechanisms. In this report, a stepwise unfolding of bovine serum albumin (BSA) with guanidine hydrochloride (GuHCl) has been investigated using Raman spectroscopy in the amide I and III regions. Two-dimensional (2D) correlation analysis has been applied to reveal information on the sequential order and the dynamic properties of interaction during the unfolding process. Raman spectral signatures in the amide I region revealed that there is no significant change in secondary structures up to 2 M concentration of GuHCl. However, 2D correlation analysis further supports the observation by inferring the strengthening of secondary structure at the expense of tertiary structure. At a higher concentration of GuHCl (2-4 M), there is an accumulation of random and ß-sheet structures that is mediated by small connecting segments of helices. It further accelerates the unfolding of helices and a complete collapse of structure. These analyses establish the ability of Raman spectroscopy to estimate the ensemble of secondary structures present in proteins. The results reveal the mechanistic details of unfolding, characterizing structure of intermediates even at high concentrations, and understanding the evolution of various secondary structures with respect to each other during unfolding. Such observations can be helpful in understanding the factors affecting the shape and size of proteins during folding/unfolding.

Challenges in application of Raman spectroscopy to biology and materials.

Raman spectroscopy has become an essential tool for chemists, physicists, biologists and materials scientists. In this article, we present the challenges in unravelling the molecule-specific Raman spectral signatures of different biomolecules like proteins, nucleic acids, lipids and carbohydrates based on the review of our work and the current trends in these areas. We also show how Raman spectroscopy can be used to probe the secondary and tertiary structural changes occurring during thermal denaturation of protein and lysozyme as well as more complex biological systems like bacteria. Complex biological systems like tissues, cells, blood serum etc. are also made up of such biomolecules. Using mice liver and blood serum, it is shown that different tissues yield their unique signature Raman spectra, owing to a difference in the relative composition of the biomolecules. Additionally, recent progress in Raman spectroscopy for diagnosing a multitude of diseases ranging from cancer to infection is also presented. The second part of this article focuses on applications of Raman spectroscopy to materials. As a first example, Raman spectroscopy of a melt cast explosives formulation was carried out to monitor the changes in the peaks which indicates the potential of this technique for remote process monitoring. The second example presents various modern methods of Raman spectroscopy such as spatially offset Raman spectroscopy (SORS), reflection, transmission and universal multiple angle Raman spectroscopy (UMARS) to study layered materials. Studies on chemicals/layered materials hidden in non-metallic containers using the above variants are presented. Using suitable examples, it is shown how a specific excitation or collection geometry can yield different information about the location of materials. Additionally, it is shown that UMARS imaging can also be used as an effective tool to obtain layer specific information of materials located at depths beyond a few centimeters.

Is chemically synthesized graphene 'really' a unique substrate for SERS and fluorescence quenching?

We demonstrate observation of Raman signals of different analytes adsorbed on carbonaceous materials, such as, chemically reduced graphene, graphene oxide (GO), multi-walled carbon nanotube (MWCNT), graphite and activated carbon. The analytes selected for the study were Rhodamine 6G (R6G) (in resonant conditions), Rhodamine B (RB), Nile blue (NBA), Crystal Violet (CV) and acetaminophen (paracetamol). All the analytes except paracetamol absorb and fluoresce in the visible region. In this article we provide experimental evidence of the fact that observation of Raman signals of analytes on such carbonaceous materials are more due to resonance effect, suppression of fluorescence and efficient adsorption and that this property in not unique to graphene or nanotubes but prevalent for various type of carbon materials.