Surface-enhanced Raman spectroscopy for forensic analysis of human semen

Identification of an unknown stain encountered at a crime scene, especially where the context of the case does not provide an indication to the identity of the stain, currently requires a number of time consuming and costly presumptive and confirmatory tests to be performed. Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic method that could allow crime scene analysts to identify unknown stains rapidly both in the laboratory and in the field. The SERS technique utilizes a laser, which interacts with molecules applied to a gold nanoparticle chip (SERS substrate) that enhances the normal Raman signal, producing a shift in energy characteristic of the vibrational modes present. Therefore, the light scattering spectrum obtained provides the analyst with a unique spectral fingerprint of the molecular components of the sample. The advantages of this SERS based method include its high sensitivity, speed, non-destructive nature, ease-of-use, minimal sample preparation requirement, portability, and multiplexing capabilities. In contrast to conventional Raman spectroscopy, SERS offers higher sensitivity resulting in small sample volumes (approximately 1 μL or less) being required for sample identification and the ability to process dilute solutions. This allows for the remaining sample to be used for other forensic tests, making the technique an ideal analytical method for use at a crime scene. vi It is hypothesized that SERS can be coupled with multivariate statistical methods, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to be established as a confirmatory technique in the forensic analysis of human body fluids. It was concluded that semen produces a spectral pattern that is consistent and readily distinct from blood, saliva, urine and vaginal fluid. In addition, this investigation identified and characterized semen from four donors, utilizing liquid semen as well as semen stains on cotton swatches and glass cover slips. Reproducibility was established by analyzing three separate SERS chips for every sample and/or solution. Ten spectra of each chip were obtained, averaged, and then compared to one another. A protocol was designed for the extraction of dried semen stains on cotton swatches and application to a SERS chip. Different extraction conditions were performed, varying both the volume of water used and the time the cutting remained submerged in the water, resulting in optimal signal from 5 μL of water for 5 minutes. Additional parameters including analysis of the perimeter of the stain and the use of saline as an extractant were examined. A second protocol for the extraction of dried semen stains from a glass cover slip was designed and tested, utilizing 1 μL of water. All experimental spectra were subjected to PCA for comparison with neat semen, and determined to be consistent. Additionally, a mixture of semen and vaginal fluid was evaluated. Visual inspection and PCA of the resulting spectra demonstrated that the mixture was a combination of both body fluids. Such samples are of particular importance in sexual assault cases. vii In summary, this preliminary study of the identification of semen using SERS demonstrates the potential for the method to be used as an investigative tool for the detection of trace amounts of human body fluids at crime scenes and within forensic laboratories. Not only is semen differentiable from other body fluids, but it is also capable of being extracted from stains and successfully identified by SER