Defense Date
2022
Document Type
Directed Research Project
First Advisor
Dr. Sarah Seashols Williams
Second Advisor
Dr. Tracey Dawson Green
Third Advisor
Dr. Joseph Reiner
Abstract
Biological mixtures which result from evidence produced in sexual assault cases are one of the most common types of mixtures observed in forensic biology casework and have contributed to evidence backlogs across the country. In the forensic community, this has highlighted the need for a more efficient cell separation method at the beginning of the DNA analysis workflow than the currently accepted method. One promising novel front-end cell separation technique is optical trapping. In heterogeneous mixtures, optical tweezers have been demonstrated to manipulate and isolate cells of interest from other cell types.
For this research, a range of 5 to 25 sperm cells were isolated in triplicate from 1:20 diluted neat semen or a male-female mixture of sperm and resuspended vaginal epithelial cells. Spermatozoa were isolated using a previously designed microfluidic device and an optical trap produced from a 700mW, 1064nm continuous wave laser. The average amount of time spent trapping per sperm cell was 2.301.17 minutes for the diluted semen samples and 2.901.40 and for the mixture samples. None of the average quantified total DNA yields were statistically different from the theoretical yields, and little or no degradation amongst trapped samples was indicated. When a simple linear regression was fit to the sample data, the number of sperm cells optically trapped explained >56% of the variability observed in the percentage of expected alleles for both sample types.
Using an updated microfluidic device design and a 5W ytterbium linearly polarized laser split into two optical traps, sperm cells were again isolated from a 1:20 diluted neat semen solution. The average time per cell spent trapping decreased to 1.38min/cell. Average total DNA yield was not significantly different from the calculated theoretical yields, and the average degradation index indicated that no DNA degradation was present in the optically trapped samples. On average 97.37 ± 2.63% of the expected alleles in the samples were observed. Additionally, resuspended vaginal epithelial cells were able to be captured with the dual optical trap setup using an attenuated laser power output.
This research has demonstrated with the use of a dual optical trap setup, a sufficient number of spermatozoa to generate a full STR profile can be captured after approximately 1 hour of optical trapping. The use of a microfluidic device minimizes the possibility for contamination events and allows for easy transfer of trapped cells to subsequent DNA analysis steps. Further, this research has demonstrated the ability to capture vaginal epithelial cells, which could eventually allow for dual capture and isolation of forensically relevant cell types on a single microfluidic device. Overall, optical trapping is a promising alternative technique for isolating forensically relevant cells of interest, such as spermatozoa, compared to the traditional differential cell lysis method.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-7-2022