*Forensics*

First, in the forensic field, emerging research has focused on the three areas of study above with fingerprints [44,134–137], blood [138,139], sweat [140,141], and one general review on the use of biocomputing in forensics [142]. These five fingerprint papers provide novel applications of biorecognition elements that can be used for future biosensing devices. These papers are centralized on the idea that people have di fferent levels of certain L-amino acids, which are related to their metabolism and di fferent traits, allowing them to be di fferentiated into groups. The blood papers use the degradation of certain enzymes in order to determine the time that a blood spot has been outside the body and to identify the age of the originator from the level of a separate enzyme. The sweat papers use the levels of di fferent compounds in sweat in order to identify sweat and to be able to provide an alternative technique for alcohol intoxication. The methodologies within these papers use di fferent biosensors, some via enzyme assays, some with chemical reactions. A generic enzymatic assay diagram can be seen in Figure 1, where specific substrates for the enzyme are used in the assay to produce byproducts, one of which is a recognition element. The last paper is a review on other trending types of fingerprint analysis such as the use of mass spectrometry, spectroscopy, nanotechnology, and combinatorial methods [44].

**Figure 1.** Generic enzymatic assay example.

A fingerprint paper from 2015 uses an enzymatic cascade utilizing L-amino acid oxidase (L-AAO) and horseradish peroxidase (HRP) in the presence of L-amino acids to produce a visible color by oxidizing the redox dye o-dianisidine, which results in a color that may be analyzed at 436 nm [134]. Due to the fact that women produce a higher amount of these amino acids than men [26–28], this assay would allow for the determination of a person's biological sex. First, the assay was performed utilizing 50 mimicked samples, 25 male and 25 female, containing 23 target amino acids in concentrations based on reported values calculated in R-project software. This resulted in the area under the curve (AUC) of a receiver operating characteristic (ROC) curve of 99%, showing a high probability for this methodology to differentiate between the two sexes. This was then repeated with three male and three female volunteers testing both their right and left thumbs in an acid extraction methodology using polyurethane film as a medium that was developed in the same article. These real samples also show definitive differentiation between the male and female prints. Different surfaces around the lab, such as a computer screen and doorknob, were also tested in this manuscript to show viability on different surfaces. An additional paper from 2016 follows the same principles as the L-AAO/HRP assay in order to find an alternative methodology to determine biological sex, this time utilizing a chemical assay [135]. This assay uses ninhydrin, which produces a colorimetric reaction with alpha-amino (α-NH2) acids [143,144]. Ninhydrin is commonly used in forensics already as it produces Ruhemann's purple as a product in the presence of all α-NH2 acids. The process in this paper is similar to the last: 50 total mimicked samples with designated concentrations of the 23 amino acids from R-project software are analyzed photometrically at 570 nm. The area under the ROC curve in this case was at 94%, showing a high probability to correctly distinguish the two sexes. After this, five male and five female volunteers were analyzed using this technique, resulting in a 91% area under the curve for authentic samples.

Following this trend, a paper from 2017 also uses a chemical assay for the determination of biological sex with the focus being on the Bradford reagent, Coomassie Brilliant Blue G-250 dye [136]. Bradford is commonly used for quantifying proteins and is less affected by reagents and nonprotein components of samples than other commonly used reagents [145]. The goal of utilizing this assay is that it only targets six specific amino acids with which to form a colorimetric complex, thus enabling a more focused approach to the determination of biological sex with the ideal being the determination of a single amino acid assay to differentiate the sexes. Contrary to the previous papers, only authentic fingerprints were sampled from 50 authentic fingerprint samples from volunteers—25 males and 25 females. This resulted in an area under the curve of the ROC graph of 99%, showing the highest probability of the three methods to correctly identify the biological sex of the fingerprint donor.

The final fingerprint paper to be examined goes one step further, by using two separate tests, each targeting a specific amino acid, in order to differentiate on the basis of biological sex [137]. The two methods used are an enzymatic cascade targeting alanine and a chemical assay that targets arginine. The alanine-targeting assay consists of a three-enzyme cascade with alanine transaminase, pyruvate

oxidase, and horseradish peroxidase [146]. In the presence of alanine in addition to the other substrates necessary for the assay, a redox dye is oxidized by HRP and can be spectrophotometrically measured. Following the previously established standard, even though the mimicked samples produced the lowest AUC of the ROC curve with a value of 82%, the authentic samples of 50 total individuals was vastly improved at 99.8%. The second technique in this research consisted of the application of the Sakaguchi Test [147], which involves α-naphthol, NaOH, and sodium hypobromite in order to form a red-colored complex. As in the previous experiments, both mimicked and authentic samples were tested, resulting in both AUCs being 100%.

In order to apply these types of biosensor techniques to other bodily fluids that are forensically relevant, blood is another focus of research. The main topic that research on blood is centered around is the estimation of the time since deposition, TSD, of a blood spot. The first paper focuses on this by measuring the levels of two biomarkers in blood, citrate kinase (CK) and alanine transaminase (ALT), which denature with the passage of time of up to 5 days [138]. The CK assay involves creatine and adenosine triphosphate as substrates for the first enzyme, CK, in a three-enzyme cascade utilizing pyruvate kinase (PK) and lactate dehydrogenase (LDH) as the other two enzymes. LDH, the third enzyme in the cascade, is the enzyme involved with production of the recognition element β-nicotinamide adenine dinucleotide from β-nicotinamide adenine dinucleotide reduced, which produces a reduction in signal at 340 nm. ALT is a two-enzyme assay that also utilizes LDH to allow for simultaneous determination. ALT recognizes the substrates alanine and α-ketoglutaric acid. By using a two-analyte system, this provides a more reliable system of determination as it has parallel markers being analyzed compared to a single marker. Building upon this, the technique outlined in the second paper uses one assay to determine not only the TSD, but also an estimation of the age of the source individual [139]. By looking at alkaline phosphatase (ALP), this research achieves both goals. This is due to the fact that ALP is a commonly used biomarker in clinical diagnostics for bone growth that relates directly to the age of the individual. To measure this, ALP converts the substrate *p*-nitrophenol phosphate into *p*-nitrophenol, which also acts as the recognition element as *p*-nitrophenol is observable at 405 nm. As ALP is a biomarker for bone growth, and that it degenerates over time when out of the body, one can obtain data about the relative age of the blood donor and the TSD up to 2 days. For the analysis of this methodology, 100 samples were prepared via the R-project software mentioned previously, split evenly between young and old, males and females. The samples provided an AUC for the ROC curve of 99% for males and 100% for the female group in di fferentiating between old and young.

The third forensic medium that is being researched currently is sweat. As previously mentioned, sweat has many forensic applications but is di fficult to identify at a crime scene. To this end, a novel methodology in order to identify sweat by use of a biosensor strip based on the detection of lactate was developed [140]. This method utilized an enzymatic assay in order to detect lactate, a major component in sweat. The assay used involved a two-enzyme cascade of lactate oxidase (LOx) and horseradish peroxidase (HRP). LOx involves the substrates lactate and oxygen, which are used to produce hydrogen peroxide. This hydrogen peroxide is then used by HRP as mentioned previously with a redox dye to produce a signal. This methodology was able to detect sweat with minimal decay for up to two weeks and at low amounts of sweat: Around 50 nL. This technique was even applied onto a paper strip modified with polystyrene for use as a field-deployable device. This optical strip provides a binary YES/NO for the presence of sweat via a color change, which is ideal for preliminary detection of sweat that can itself be analyzed further. Additionally, sweat was further examined in a noninvasive testing methodology for ethanol sensing on the surface of one's skin [141]. This conceptualizes an alternative method to breathalyzers by relying on an enzymatic assay involving alcohol oxidase (AOx) and HRP that is quantified not only by UV-Vis spectrophotometry but also an optical camera. AOx uses ethanol and oxygen as substrates to produce hydrogen peroxide, which is used by HRP as mentioned previously. This research shows that there is a correlation between both techniques and the currently used breathalyzer. The data were achieved from a 26-volunteer drinking study with people of di fferent ages, biological sexes, and food habits. The sweat samples were obtained through pilocarpine electrophoresis similar to the Gibson and Cooke method [148], which allowed the sweat to be collected in gauze pads and analyzed. A minimum of 3 μL of sweat was required for this method.

These advances with biosensors in the field of forensics have produced a viable way for investigators to receive some information to pursue leads if DNA evidence is backlogged or not applicable using sweat and blood evidence found at the crime scene. Multiple enzymatic assays were developed for the di fferentiation of biological sex of an individual from fingerprint content, enabling an alternative or additional analysis for fingerprints depending on the clarity of the print for pictorial analysis. Blood was examined, and provides a viable methodology in order to show the time since deposition, TSD, and also an estimated age of the originator of the blood spot. Additionally, a field-deployable testing strip was developed for the determination of sweat, supplying clarity for a di fficult-to-detect bodily fluid at crime scenes. Sweat was also tested in a laboratory setting for an alternative to breathalyzers for the detection and quantification of alcohol in sweat utilizing enzymatic assays and a colorimetric response. In the future, di fferent drugs and illicit substances, such as THC, in addition to a more broad analysis of metabolites characteristic to certain habits or biological features can be examined for their use in forensics for providing a more deterministic and rapid analysis for forensic and law enforcement personnel in a sweeping suite of biosensor devices.
