In Situ Stability Test for mRNA Vaccines Based on Deep-UV Resonance Raman Spectroscopy.

Deep-UV resonance Raman spectroscopy has been shown to offer great potential for probing the in situ stability of mRNA vaccines. In this study, a vaccine model was subjected to controlled degradation using RNase A or through aging at room temperature. The degradation of mRNA was confirmed by using a cell transfection test and by gel electrophoresis. Under both settings, DUVRR spectroscopy successfully revealed the mRNA degradation signs of the vaccine model.

Raman Spectroscopy for the Time since Deposition Estimation of a Menstrual Bloodstain.

Forensic chemistry plays a crucial role in aiding law enforcement investigations by applying analytical techniques for the analysis of evidence. While bloodstains are frequently encountered at crime scenes, distinguishing between peripheral and menstrual bloodstains presents a challenge. This is due to their similar appearance post-drying. Raman spectroscopy has emerged as a promising technique capable of discriminating between the two types of bloodstains, offering invaluable probative information. Moreover, estimating the time since deposition (TSD) of bloodstains aids in crime scene reconstruction and prioritizing what evidence to collect. Despite extensive research focusing on TSD estimations, primarily in peripheral bloodstains, a crucial gap exists in determining the TSD of menstrual bloodstains. This study demonstrates how Raman spectroscopy effectively analyzes biological samples like menstrual blood, showing similar aging patterns to those of peripheral blood and provides proof-of-concept models for determining the TSD of menstrual blood. While this work shows promising results for creating a universal model for bloodstain age determination, further testing with more donors needs to be conducted before the implementation of this method into forensic practice.

Diagnosing COVID-19 in nasopharyngeal secretion through Raman spectroscopy: a feasibility study.

Since the beginning of the COVID-19 pandemic, the scientific community has sought to develop fast and accurate techniques for detecting the SARS-CoV-2 virus. Raman spectroscopy is a promising technique for diagnosing COVID-19 through serum samples. In the present study, the diagnosis of COVID-19 through nasopharyngeal secretion has been proposed. Raman spectra from nasopharyngeal secretion samples (15 Control, negative and 12 COVID-19, positive, assayed by immunofluorescence antigen test) were obtained in triplicate in a dispersive Raman spectrometer (830 nm, 350 mW), accounting for a total of 80 spectra. Using principal component analysis (PCA) the main spectral differences between the Control and COVID-19 samples were attributed to N and S proteins from the virus in the COVID-19 group. Features assigned to mucin (serine, threonine and proline amino acids) were observed in the Control group. A binary model based on partial least squares discriminant analysis (PLS-DA) differentiated COVID-19 versus Control samples with accuracy of 91%, sensitivity of 80% and specificity of 100%. Raman spectroscopy has a great potential for becoming a technique of choice for rapid and label-free evaluation of nasopharyngeal secretion for COVID-19 diagnosis.

Spontaneous Refolding of Amyloid Fibrils from One Polymorph to Another Caused by Changes in Environmental Hydrophobicity.

Here, we report a new phenomenon in which lysozyme fibrils formed in a solution of acetic acid spontaneously refold to a different polymorph through a disassembled intermediate upon the removal of acetic acid. The structural changes were revealed and characterized by deep-UV resonance Raman spectroscopy, nonresonance Raman spectroscopy, intrinsic tryptophan fluorescence spectroscopy, and atomic force microscopy. A PPII-like structure with highly solvent-exposed tryptophan residues predominates the intermediate aggregates before refolding to polymorph II fibrils. Furthermore, the disulfide (SS) bonds undergo significant rearrangements upon the removal of acetic acid from the lysozyme fibril environment. The main SS bond conformation changes from gauche-gauche-trans in polymorph I to gauche-gauche-gauche in polymorph II. Changing the hydrophobicity of the fibril environment was concluded to be the decisive factor causing the spontaneous refolding of lysozyme fibrils from one polymorph to another upon the removal of acetic acid. Potential biological implications of the discovered phenomenon are discussed.

Surface-enhanced Raman spectroscopy for drug discovery: peptide-RNA binding.

The ever-growing demand for new drugs highlights the need to develop novel cost- and time-effective techniques for drug discovery. Surface-enhanced Raman spectroscopy (SERS) is an emerging ultrasensitive and label-free technique that allows for the efficient detection and characterization of molecular interactions. We have recently developed a SERS platform for detecting a single protein molecule linked to a gold substrate (Almehmadi et al. Scientific Reports 2019). In this study, we extended the approach to probe the binding of potential drugs to RNA targets. To demonstrate the proof of concept, two 16-amino acid residue peptides with close primary structures and different binding affinities to the RNA CUG repeat related to myotonic dystrophy were tested. Three-microliter solutions of the RNA repeat with these peptides at nanomolar concentrations were probed using the developed approach, and the binding of only one peptide was demonstrated. The SER spectra exhibited significant fluctuations along with a sudden strong enhancement as spectra were collected consecutively from individual spots. Principal component analysis (PCA) of the SER spectral datasets indicated that free RNA repeats could be differentiated from those complexed with a peptide with 100% accuracy. The developed SERS platform provides a novel opportunity for label-free screening of RNA-binding peptides for drug discovery. Schematic representation of the SERS platform for drug discovery developed in this study.

Discrimination of menstrual and peripheral blood traces using attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy and chemometrics for forensic purposes.

Body fluid traces can provide highly valuable clues in forensic investigations. In particular, bloodstains are a common occurrence in criminal investigation, and the discrimination of menstrual and peripheral blood is a crucial step for casework involving rape and sexual assault. Most of the current protocols require the detection of characteristic menstrual blood components using sophisticated procedures that need to be performed in a laboratory. The present study uses attenuated total reflection Fourier transform-infrared (ATR FT-IR) spectroscopy as a nondestructive technique for discriminating menstrual and peripheral blood traces. This method incorporates statistical analysis and was evaluated by internal and external validation testing. A partial least squares discriminant analysis (PLSDA) classification model was created for differentiating the two types of blood in a binary manner. Excellent separation between menstrual and peripheral blood samples was achieved during internal validation. External validation resulted in 100% accuracy for predicting a sample as peripheral or menstrual blood. This study demonstrates that ATR FT-IR spectroscopy combined with chemometrics is a reliable approach for rapid and nondestructive discrimination of menstrual and peripheral bloodstains. It offers a significant advantage to forensic science due to the availability of portable instruments and the potential for bloodstain analysis at a crime scene. Graphical abstract.

Towards development of a novel universal medical diagnostic method: Raman spectroscopy and machine learning.

Many problems exist within the myriad of currently employed screening and diagnostic methods. Further, an incredibly wide variety of procedures are used to identify an even greater number of diseases which exist in the world. There is a definite unmet clinical need to improve diagnostic capabilities of these procedures, including improving test sensitivity and specificity, objectivity and definitiveness, and reducing cost and invasiveness of the test, with an interest in replacing multiple diagnostic methods with one powerful tool. There has been a recent surge in the literature which focuses on utilizing Raman spectroscopy in combination with machine learning analyses to improve diagnostic measures for identifying an assortment of diseases, including cancers, viral and bacterial infections, neurodegenerative and autoimmune disorders, and more. This review highlights the work accomplished since 2018 which focuses on using Raman spectroscopy and machine learning to address the need for better screening and medical diagnostics in all areas of disease. A critical evaluation considers both the benefits and obstacles of utilizing the method for universal diagnostics. It is clear based on the evidence provided herein Raman spectroscopy in combination with machine learning provides the first glimmer of hope for the development of an accurate, inexpensive, fast, and non-invasive method for universal medical diagnostics.

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

A Novel Two-Step Method for the Detection of Organic Gunshot Residue for Forensic Purposes: Fast Fluorescence Imaging Followed by Raman Microspectroscopic Identification.

Gunshot residue (GSR) is potentially key evidence during a criminal investigation of a shooting accident. Current standardized forensic science methods target the detection of inorganic GSR (IGSR). In this proof-of-concept study, a new two-step method for the detection and identification of organic GSR (OGSR) is proposed. This method utilizes highly sensitive fluorescence hyperspectral imaging of a sample area to detect potential GSR particles, followed by confirmatory identification of the detected particles using Raman microspectroscopy. In this study, two different GSR samples on adhesive tape substrates were created. One sample was made by manually placing a known amount of OGSR particles onto an adhesive tape substrate. The second sample mimicked a real crime scene situation and had an unknown number of GSR particles mounted onto an adhesive tape substrate using a most common tape-lifting procedure for the recovery of GSR from the skin of a suspect and other surfaces. These two samples were subjected to the developed two-step analysis method. It was found that this method was accurately able to detect and identify all OGSR particles. Representative spectra of OGSR particles showed characteristic Raman peaks at 850 cm-1, 1287 cm-1, and 2970 cm-1. This methodology offers a promising means to meet current needs within the framework of GSR analysis by providing a way to accurately detect and identify OGSR.

Phenotype Profiling for Forensic Purposes: Determining Donor Sex Based on Fourier Transform Infrared Spectroscopy of Urine Traces.

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special importance to the modern criminal investigation as a source of individualizing DNA evidence. However, individual identification of a urine donor is generally difficult because of the small amount of DNA. Therefore, the development of an innovative method to provide phenotype information about the urine donor-including sex-is highly desirable. In this study, we developed a multivariate discriminant model for the ATR FT-IR spectra of dry urine to identify the donor sex. Rigorous selection of significant wavenumbers on the spectrum using a genetic algorithm enabled superb discrimination performance for the model and conclusively indicated a chemical origin for donor sex differences, which was supported by physiological knowledge. Although further investigations need to be conducted, this proof-of-concept study demonstrates the great potential of the developed methodology for phenotype profiling based on the analysis of urine traces.

Ultraviolet Resonance Raman Spectroscopic Markers for Protein Structure and Dynamics.

UV resonance Raman (UVRR) spectroscopy is a powerful tool for investigating the structure of biological molecules, such as proteins. Numerous UVRR spectroscopic markers that provide information on the structure and environment of the protein backbone and of amino acid side chains have recently been discovered. Combining these UVRR markers with hydrogen-deuterium exchange and advanced statistics is a powerful tool for studying protein systems, including the structure and formation mechanism of protein aggregates and amyloid fibrils. These techniques allow crucial new insights into the structure and dynamics of proteins, such as polyglutamine peptides, which are associated with 10 different neurodegenerative diseases. Here we summarize the spectroscopic structural markers recently developed and the important insights they provide.

Raman microspectroscopic mapping as a tool for detection of gunshot residue on adhesive tape.

Our research group previously reported a novel method for the detection of gunshot residue (GSR) via tape lifting combined with Raman microspectroscopic mapping and multivariate analysis. This initial study achieved proof of concept for this approach. Here, we report validation studies which investigate the reproducibility/ruggedness and specificity of the approach. Raman mapping for GSR detection on adhesive tape was performed on an independent Raman microscope, not used to generate the training set. These independent spectra were classified against the original training dataset using support vector machine discriminant analysis (SVM-DA). The resulting classification rates of 100% illustrate the reproducibility of the technique, its independence upon a specific instrument and provide an external validation for the approach. Additionally, the same procedure for GSR collection (tape lifting) was performed to collect samples from environmental sources, which could potentially provide false-positive assignments for current GSR analysis techniques. Thus, particles associated with automotive mechanics were collected. Automotive brake and tire materials are often composed of the heavy metals lead, barium, and antimony, which are the key elements targeted by current GSR detection technique. It was determined that Raman spectroscopic analysis was not susceptible to misclassifications from these samples. Results from these validation experiments illustrate the great potential of Raman microspectroscopic mapping used with tape lifting as a viable complimentary tool to current methodologies for GSR detection. Furthermore, current methodologies are not well-developed for automated organic GSR detection. Illustrated here, Raman microscoptrosocpic mapping has the potential for the automatic identification of organic GSR. Graphical abstract ᅟ.

Purple Fibrils: A New Type of Protein Chromophore.

A purple color is formed during the fibrillation of lysozyme, a well-studied protein lacking a prosthetic group. The application of Raman spectroscopy, electron paramagnetic resonance and UV-vis absorption spectroscopy indicates the formation of a sulfur∴π-bonded radical cation due to the methionine-phenylalanine interaction, which is consistent with a small molecule model reported in the literature. A purple chromophore with characteristic 550 nm absorption is formed due to a specific orientation of the sulfur-centered radical cation and a phenyl ring stabilized by the fibril framework. A specific fibril conformation and the resulting formation of the chromophore are controlled reversibly by varying the pH. This is the first known example of a side chain self-assembled chromophore formed due to protein aggregation.

Determining Gender by Raman Spectroscopy of a Bloodstain.

The development of novel methods for forensic science is a constantly growing area of modern analytical chemistry. Raman spectroscopy is one of a few analytical techniques capable of nondestructive and nearly instantaneous analysis of a wide variety of forensic evidence, including body fluid stains, at the scene of a crime. In this proof-of-concept study, Raman microspectroscopy was utilized for gender identification based on dry bloodstains. Raman spectra were acquired in mapping mode from multiple spots on a bloodstain to account for intrinsic sample heterogeneity. The obtained Raman spectroscopic data showed highly similar spectroscopic features for female and male blood samples. Nevertheless, support vector machines (SVM) and artificial neuron network (ANN) statistical methods applied to the spectroscopic data allowed for differentiating between male and female bloodstains with high confidence. More specifically, the statistical approach based on a genetic algorithm (GA) coupled with an ANN classification showed approximately 98% gender differentiation accuracy for individual bloodstains. These results demonstrate the great potential of the developed method for forensic applications, although more work is needed for method validation. When this method is fully developed, a portable Raman instrument could be used for the infield identification of traces of body fluids and to obtain phenotypic information about the donor, including gender and race, as well as for the analysis of a variety of other types of forensic evidence.

Race Differentiation Based on Raman Spectroscopy of Semen Traces for Forensic Purposes.

Several novel methods to determine externally visible characteristics of body fluid donors have been developed in recent years. These tests can help forensic investigators make predictions about the appearance of a suspect or victim, such as their sex, race, hair color, or age. While their potential benefit is undeniable, these methods destroy the physical evidence in the process. Raman spectroscopy has recently been used as a nondestructive technique to test for many of these characteristics. Here, we present the results from a study to determine the race of semen donors. Using Raman spectroscopy and multivariate data analysis, we were able to build a statistical model that accurately identified the race of all 18 semen donors in the calibration data set, as well as seven additional external validation donors. These results demonstrate Raman spectroscopy's potential to differentiate Caucasian and Black semen donors using chemometrics.

Identification of individual red blood cells by Raman microspectroscopy for forensic purposes: in search of a limit of detection.

Traces of body fluids can be present at a variety of crime scenes. It is important that forensic investigators have a reliable and nondestructive method of identifying these traces. Of equal importance is establishing the limitations of any method in use, including its detection limit. We have previously reported on the use of Raman microspectroscopy and multivariate data analysis to identify and differentiate body fluids. While many studies use serial dilutions to establish limits of detection, we utilized a different approach and demonstrated that a single red blood cell is sufficient to be correctly identified as blood. The experimental Raman spectra of individual red blood cells were loaded into the previously reported models for body fluid identification, and all were correctly classified as peripheral blood. These results demonstrate that our model can be used to identify peripheral blood, even if there is only a single red blood cell present. Furthermore, a single red blood cell is 5000× smaller than the amount of peripheral blood required to perform DNA analysis in a modern crime laboratory. This means that if a bloodstain is large enough for DNA analysis, Raman microspectroscopy should be able to make a positive identification. Considering that the sample analysis reported here was carried out with a different instrument, not the one used for the previously reported method development, these results also represent a form of method validation. The model's ability to correctly classify spectra acquired on a different instrumental platform is crucial in preparing it for practical application. Graphical Abstract Peripheral blood is of great interest in forensic sciences. While many tests are available for the identification of peripheral blood at a crime scene, most are presumptive and destructive. Here we present results that show our new, nondestructive method can identify peripheral blood using as little as a single red blood cell.

Origin of enhanced VCD in amyloid fibril spectra: Effect of deuteriation and pH.

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D2 O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.

Sex Determination Based on Raman Spectroscopy of Saliva Traces for Forensic Purposes.

The forensic analysis of body fluids has made great strides in recent years. Body fluids can easily be identified, and DNA analysis can be used to link a stain found at a crime scene to a specific person. When no reference DNA profile is available and the recovered DNA does not yield a match in a database, it would be incredibly useful if the evidence could still provide investigators with useful information. Biocatalytic and immunoassays can be used to determine a donor's sex, race, and other phenotypic characteristics. However, these tests depend on chemical reactions and are destructive to the sample. Here, we used Raman spectroscopy and multivariate data analysis to develop a nondestructive technique that could be used at a crime scene to determine the sex of a saliva donor. Our internally cross-validated classification model correctly identified 44 (92%) of the 48 donors used for model training. Subsequent external validation correctly identified 11 (92%) of the 12 donors saved for testing. This proof-of-concept study demonstrates the value of Raman spectroscopy as a forensic tool, and indicates that it can be used to elucidate phenotypic information about a body fluid donor. Future studies will expand to other body fluids and additional donor characteristics, such as race and age.

Race Differentiation by Raman Spectroscopy of a Bloodstain for Forensic Purposes.

Bearing in mind forensic purposes, a nondestructive and rapid method was developed for race differentiation of peripheral blood donors. Blood is an extremely valuable form of evidence in forensic investigations so proper analysis is critical. Because potentially miniscule amounts of blood traces can be found at a crime scene, having a method that is nondestructive, and provides a substantial amount of information about the sample, is ideal. In this study Raman spectroscopy was applied with advanced statistical analysis to discriminate between Caucasian (CA) and African American (AA) donors based on dried peripheral blood traces. Spectra were collected from 20 donors varying in gender and age. Support vector machines-discriminant analysis (SVM-DA) was used for differentiation of the two races. An outer loop subject-wise cross-validation (CV) method evaluated the performance of the SVM classifier for each individual donor from the training data set. The performance of SVM-DA, evaluated by the area under the curve (AUC) metric, showed 83% probability of correct classification for both races, and a specificity and sensitivity of 80%. This preliminary study shows promise for distinguishing between different races of human blood. The method has great potential for real crime scene investigation, providing rapid and reliable results, with no sample preparation, destruction, or consumption.