Defense Date


Document Type

Directed Research Project

First Advisor

Catherine Cupples Connon, PhD

Second Advisor

Joseph Turner, PhD

Third Advisor

Emanuele Alves, PhD

Fourth Advisor

Eric Hazelrigg, MS


Blood is one of the most important types of evidence across many forensic investigations. To promote the authenticity of blood evidence, a colorimetric assay for the detection of ethylenediaminetetraacetic acid (EDTA) in blood was previously developed. The colorimetric test in conjunction with UV-Visible spectrophotometry was coined the RED-BLEU (Reverse EDTA Detection in Blood Using EBT and UV-Vis) assay. During early testing it was demonstrated that the EDTA peak is not visible when blood with EDTA is analyzed on the NanoDrop ND-2000 UV Spectrophotometer; this is suspected to be due to interference from blood proteins. The interfering blood peak is very tall and wide, masking any peaks that may be present in the same area, including the wavelength of the expected EDTA peak. If an effective sample cleanup method is established, UV-vis can be paired with the colorimetric test to increase the accuracy of results from the RED-BLEU assay. Five protein precipitation/digestion methods were tested for integration into the RED-BLEU assay: ammonium sulfate, trichloroacetic acid, sodium hydroxide, Proteinase K, and trypsin. These methods were evaluated for their compatibility with the colorimetric test and the visibility of the EDTA peak in the UV-Vis spectra of samples. Ammonium sulfate, sodium hydroxide, Proteinase K, and trypsin all interfered with the color test results to some degree. Treatment with trichloroacetic acid did maintain expected color test results, however, distinguishing samples with and without EDTA via UV-Vis was not possible with this method. Ammonium sulfate and trypsin also did not reduce interference in the UV-Vis spectra enough to distinguish between samples with and without EDTA. Sodium hydroxide was somewhat successful at helping distinguish samples with and without EDTA with UV-Vis based on the wavelength of peak maximum between 190 and 200 nm. Proteinase K was the most promising method for reducing interference in the UV-Vis spectra. To prevent interference with the color results, Proteinase K was incorporated into the RED-BLEU assay after the color test was performed. Recommended parameters for the use of Proteinase K were tested to determine the necessary concentration, incubation time, and incubation temperature for integration into the RED-BLEU assay. Overall, both Proteinase K and sodium hydroxide should be revisited for further optimization to determine which method best supports the goal of a fast, affordable presumptive test for EDTA detection in blood.


© The Author(s)

Is Part Of

VCU Master of Science in Forensic Science Directed Research Projects

Date of Submission


Available for download on Saturday, May 08, 2027