Alkaline Phosphatase for Estimating the Time since Deposition of Blood Fingerprints by Scanning Electrochemical Microscopy.

Blood is one of the most frequent and valuable traces encountered at crime scenes, where knowing the time since deposition (TSD) of bloodstains tremendously assists forensic experts to screen out crime-related evidence and aids in the reconstruction of the event sequence. Although increasing proof-of-concept methodologies for investigating the TSD of bloodstains have been reported, there is still no accepted strategy in forensic practice as the aging mechanism involves complex components, leading to the inaccuracy of the estimation results. Herein, an endogenous biomarker of alkaline phosphatase (ALP) was chosen to investigate the TSD by scanning electrochemical microscopy (SECM). Results demonstrate that the ALP activity acquired via SECM lateral scan assay exhibited a clear decrease over time, and a similar trend was observed on both poly(vinylidene fluoride) (PVDF) membrane and glass, with the aging kinetics on PVDF membrane being faster than glass. By means of quantitatively calculating the flux of generated p-aminophenol (PAP), we established the aging curve and realized the TSD estimation of blood fingerprints (BFPs) that was unable to be distinguished via optical measurements. Intriguingly, the as-obtained estimation accuracy ranged from 74.6 to 93.7%, proving the possibility of using an ALP biomarker and SECM. More appealingly, the predicted TSDs were capable of accurately differentiating the deposition sequence of overlapping BFPs, which was hardly achieved by optical means. Therefore, this proof-of-concept strategy demonstrates the value of SECM as a forensic tool and opens possibilities for revealing multidimensional information about crime.