Attenuated Total Reflectance-FT-IR Imaging for Rapid and Automated Detection of Gunshot Residue

An alternative approach for the nondestructive, rapid and selective detection of gunshot residue (GSR) was investigated. A cloth substrate containing GSR particles expelled during a firearm discharge was used as an analog for the clothing of a shooting victim or a suspect discharging a firearm. An established and efficient procedure for GSR collection (tape lifting) was utilized to recover GSR particles from the cloth substrate. Microscopic-attenuated total reflectance (ATR) Fourier transform (FT) infrared (IR) spectroscopic imaging rapidly and automatically scanned large areas of the tape collection substrate and detected varying morphologies (microscopic and macroscopic) and chemical compositions (organic and inorganic) of GSR. The “spectroscopic fingerprint” of each GSR type provided unique virbrational modes, which were not characteristic of the tape collection substrate or the cloth debris which was also recovered. ATR images (maps) targeted the detection of these unique chemical markers over the mapped area. The hues of the ATR images were determined by the intensity of the signal for the chemical marker of each analyte. The spatial resolution of the technique was determined to be 4.7 μm. Therefore, all GSR particles sized 4.7 μm or larger will be resolved and detected on the tape substrate using micro-ATR imaging

Differentiating Donor Age Groups Based on Raman Spectroscopy of Bloodstains for Forensic Purposes

Developments in analytical chemistry technologies and portable instrumentation over the past decade have contributed significantly to a variety of applications ranging from point of care testing to industrial process control. In particular, Raman spectroscopy has advanced for analyzing various types of evidence for forensic purposes. Extracting phenotypic information (e.g., sex, race, age, etc.) from body fluid traces is highly desirable for criminal investigations. Identifying the chronological age (CA) of a blood donor can provide significant assistance to detectives. In this proof-of-concept study, Raman spectroscopy and chemometrics have been used to analyze blood from human donors, and differentiate between them based on their CA [i.e., newborns (CA of <1 year), adolescents (CA of 11–13 years), and adults (CA of 43–68 years)]. A support vector machines discriminant analysis (SVMDA) model was constructed, which demonstrated high accuracy in correctly predicting blood donors’ age groups where the lowest cross-validated sensitivity and specificity values were 0.96 and 0.97, respectively. Overall, this preliminary study demonstrates the high selectivity of Raman spectroscopy for differentiating between blood donors based on their CA. The demonstrated capability completes our suite of phenotype profiling methodologies including the determination of sex and race. CA determination has particular importance since this characteristic cannot be determined through DNA profiling unlike sex and race. When completed, the developed methodology should allow for phenotype profiling based on dry traces of body fluids immediately at the scene of a crime. The availability of this information within the first few hours since the crime discovery could be invaluable for the investigation

Attenuated Total Reflectance-FT-IR Spectroscopy for Gunshot Residue Analysis: Potential for Ammunition Determination

The ability to link a suspect to a particular shooting incident is a principal task for many forensic investigators. Here, we attempt to achieve this goal by analysis of gunshot residue (GSR) through the use of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FT-IR) combined with statistical analysis. The firearm discharge process is analogous to a complex chemical process. Therefore, the products of this process (GSR) will vary based upon numerous factors, including the specific combination of the firearm and ammunition which was discharged. Differentiation of FT-IR data, collected from GSR particles originating from three different firearm–ammunition combinations (0.38 in., 0.40 in., and 9 mm calibers), was achieved using projection to latent structures discriminant analysis (PLS-DA). The technique was cross (leave-one-out), both internally and externally, validated. External validation was achieved via assignment (caliber identification) of unknown FT-IR spectra from unknown GSR particles. The results demonstrate great potential for ATR-FT-IR spectroscopic analysis of GSR for forensic purposes

Structural Organization of Insulin Fibrils Based on Polarized Raman Spectroscopy: Evaluation of Existing Models

Many different proteins undergo misfolding and self-assemble into amyloid fibrils, resulting in a range of neurodegenerative diseases. The limitations of conventional methods of structural biology for fibril characterization have led to the use of polarized Raman spectroscopy for obtaining quantitative structural information regarding the organization of amyloid fibrils. Herein, we report the orientation of selected chemical groups and secondary structure elements in aligned insulin fibrils, including β-sheets, which possess a high level of orientation in the cross-β core, and α-helices in the disordered portions of the fibrils. Strong orientation of disulfide bonds in amyloid fibrils was also revealed, indicating their association with the fibril core. The determined orientation of chemical groups provides strong constraints for modeling the overall structure of amyloid fibrils, including the core and disordered parts. The developed methodology allows for the validation of structural models proposed in the literature for amyloid fibrils. Specifically, the polarized Raman data obtained herein strongly agreed with two insulin fibril models (Jiménez et al., <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2002</b>, <i>99</i>, 9196–9201 and Ivanova et al., <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2009</b>, <i>106</i>, 18990–18995) yet revealed significant qualitative and quantitative differences. This work demonstrates the great potential of polarized Raman spectroscopy for structural characterization of anisotropic biological species

Raman Spectroscopy of Blood for Species Identification

The species identification of a blood stain is an important and immediate challenge for forensic science, veterinary purposes, and wildlife preservation. The current methods used to identify the species of origin of a blood stain are limited in scope and destructive to the sample. We have previously demonstrated that Raman spectroscopy can reliably differentiate blood traces of human, cat, and dog (Virkler et al. Anal. Chem. 2009, 81, 7773−7777) and, most recently, built a binary model for differentiating human vs animal blood for 11 species integrated with human existence (McLaughlin et al. Forensic Sci. Int. 2014, 238, 91−95). Here we report a satisfactory classification of blood obtained from 11 animal classes and human subjects by statistical analysis of Raman spectra. Classification of blood samples was achieved according to each sample’s species of origin, which enhanced previously observed discrimination ability. The developed approach does not require the knowledge of a specific (bio)­chemical marker for each individual class but rather relies on a spectroscopic statistical differentiation of various components. This approach results in remarkable classification ability even with intrinsically heterogeneous classes and samples. In addition, the obtained spectroscopic characteristics could potentially provide information about specific changes in the (bio)­chemical composition of samples, which are responsible for the differentiation

Structure and Composition of Insulin Fibril Surfaces Probed by TERS

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobson’s model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196–9201). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization

Deconstruction of Stable Cross-Beta Fibrillar Structures into Toxic and Nontoxic Products Using a Mutated Archaeal Chaperonin

Our group recently determined that a mutant archaeal chaperonin (Hsp 60) exhibited substantially enhanced protein folding activity at low temperatures and was able to deconstruct refractory protein aggregates. ATP dependent conversion of fibril structures into amorphous aggregates was observed in insulin amyloid preparations (Kurouski et al. <i>Biochem. Biophys. Res. Commun.</i> 2012). In the current study, mechanistic insights into insulin fibril deconstruction were obtained by examination of early stage complexes between Hsp60 and fibrils in the absence of ATP. Activity of the Hsp60 was significantly curtailed without ATP; however, some fibril deconstruction occurred, which is consistent with some models of the folding cycle that predict initial removal of unproductive protein folds. Chaperonin molecules adsorbed on the fibril surface and formed chaperonin clusters with no ATP present. We propose that there are specific locations on the fibril surface where chaperonin can unravel the fibril to release short fragments. Spontaneous coagulation of these fibril fragments resulted in the formation of amorphous aggregates without the release of insulin into solution. The addition of ATP significantly increased the toxicity of the insulin fibril-chaperonin reaction products toward mammalian cells

Disulfide bonds preserve their conformation upon insulin fibrillation.

<p>Raman spectra of native insulin (red) and insulin fibrils (black) have a peak at 510 cm⁻¹, corresponding to the gauche-gauche-gauche (g-g-g) conformation of disulfide bonds (schematically represented in the inset).</p

The majority of H/D exchange-protected amino acids are located in the B-chain of insulin.

<p>The amino acids in the 3D structure (A) and primary sequence of insulin fibrillar monomer (B) are colored according to the following degree of H/D exchange protection: yellow, I_D2O/I_H2O≥0.75; red, 0.75>I_D2O/I_H2O≥0.675; and blue, 0.675>I_D2O/I_H2O≥0.60. Gray (in the 3D structure) and white colors (in the primary sequence) indicate unprotected (exchanged) amino acids. (C) 2D ¹H,¹H-TOCSY spectrum of insulin fibrillar monomer in DMSO and 0.05% TFA at 30°C after a 7-day D₂O exchange at room temperature. NMR data were acquired at room temperature on a Bruker Avance II 500 MHz NMR spectrometer equipped with a cryoprobe.</p

The α-helix and β-sheet character of simulated insulin monomer and dimer.

<p>The α-helix and β-sheet character of simulated insulin monomer and dimer.</p