Development of Enzyme- and Protein-Functionalized Alginate Hydrogels with Luminol Catalytic Functionality for Use as Forensic Blood Substitutes

Forensic blood substitutes (FBS) are materials that are synthetic alternatives to whole blood for research and training purposes in the forensic sciences. One of many desirable features of an FBS is its ability to mimic latent (invisible) bloodstains that can be enhanced with common searching reagents like luminol. In this work, two alginate-based biomolecule-functionalized hydrogels were developed and evaluated for their ability to catalyze the oxidation of luminol like whole blood. A protein-based FBS functionalized with hemoglobin (Hb; AlgXHb) and enzyme-based FBS functionalized with horseradish peroxidase (HRP; AlgXHRP) were developed and characterized using UV-vis spectroscopy and rheology. The enhanced materials were compared to the chemiluminescence of whole bovine blood dilution standards (neat to 1:1,000,000). The AlgXHb and AlgXHRP materials, both as thin films and microparticles, produced chemiluminescence when enhanced with luminol, however with varying reaction kinetics and emission intensities. AlgXHb produced chemiluminescence comparable to 1:1000 whole blood dilution, with a short half-life (∼17 s) and reaction kinetics which mimic latent blood. AlgXHRP chemiluminescence was comparable to a 1:10,000 whole blood dilution, with an extended half-life (∼60 s), and differing reaction kinetics resulting in prolonged emission. The AlgXHb material was further diluted to test its working capacity as mimetic latent bloodstain evidence (shoeprint transfer patterns) and as a luminol control material (deposited on cotton applicator swabs). Both materials were compared with and validated to latent bloodstain standards and offer applications in using a functional alternative to whole blood as an on-scene luminol reagent test as well as a training material for forensic practitioners.