Fingerprint Analysis: Moving Toward Multiattribute Determination via

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Fingerprint Analysis: Moving Toward Multiattribute Determination via Individual Markers Erica Brunelle, Crystal Huynh, Eden Alin, Morgan Eldridge, Anh Minh Le, Lenka Halámková, and Jan Halámek Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04206 • Publication Date (Web): 02 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Analytical Chemistry

Fingerprint Analysis: Moving Toward Multi-attribute Determination via Individual Markers Authors: Erica Brunelle1, Crystal Huynh1, Eden Alin1, Morgan Eldridge1, Anh Minh Le1, Lenka Halámková1, and Jan Halámek1* Affiliations: 1 Dept. of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., 12222, NY *

Correspondence to: [email protected]

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Abstract Forensic science will be forever revolutionized if law enforcement can identify personal attributes of a person of interest solely from a fingerprint. For the past 2 years, the goal of our group was to establish a way to identify originator attributes, specifically biological sex, from a single analyte. To date, an enzymatic assay and two chemical assays have been developed for the analysis of multiple analytes. In this manuscript, two additional assays have been developed. This time, however, the assays utilize only one amino acid each. The enzymatic assay targets alanine and employs alanine transaminase (ALT), pyruvate oxidase (POx), and horseradish peroxidase (HRP). The other, a chemical assay, is known as the Sakaguchi test and targets arginine. It is important to note that alanine is the fifth highest amino acid concentration in fingerprint content and arginine is the lowest. Both assays proved to be capable of accurately differentiating between male and female fingerprints, regardless of their respective average concentration. The ability to target a single analyte will transform forensic science as each originator attribute can be correlated to a different analyte. This would then lead to the possibility of identifying multiple attributes from a single fingerprint sample. Ultimately, this would allow for a profile of a person of interest to be established without the need for time consuming lab processes. Introduction In modern criminology, biological traces such as fingerprints and DNA found at crime scenes represent important leads in the identification of possible suspects. Chemical and biochemical techniques utilized for the analysis of biological traces at crime scenes are the main scientific support of criminal investigations and subsequent prosecutions.1 Fingerprints, in particular, are samples of biological origin analogous to other body fluids such as blood. Over the past two years, our group, among others,2-6 has begun to investigate the potential for fingerprints to provide more information about the originator. This has been accomplished using alternative approaches to traditional biochemical and chemical techniques have also been implemented. It has been established that the contents of fingerprints are produced by multiple hormonebased control mechanisms7 and thus, are a function of physical properties such as biological sex, age, ethnicity, or health status.8-16 In our recently published work, 17,18 we have demonstrated that fingerprints have the potential to provide much more information other than just providing an image. These manuscripts have encompassed multi-analyte enzymatic and chemical assays, both of which focused on the detection of 23 amino acids – 20 natural and 3 unnatural – where the overall concentration of amino acids is correlated to the biological sex of the fingerprint originator with females having the higher concentration. However, our group’s intentions are to establish a method where only one amino acid corresponds to only one originator attribute. Multi-analyte assays, such as the ones previously developed, are not completely reliable for attribute determination because more than one attribute can ultimately effect the output, thus convoluting the intentions of the assay. Prior to investigating single analyte methods, we optimized the a chemical assay19 for biological sex identification with the sole purpose of exploring the effect of decreasing the number of amino acid targets, while not compromising the integrity of the concept. This assay19 was ultimately successful in identifying biological sex from fingerprints even though the amino acid target population was drastically decreased from 23 to 6. These results brought us closer to our goal of establishing the use of one amino acid for one originator attribute so that eventually, one fingerprint can be used to identify multiple attributes via individual markers (analytes). Initially we went back to our original platform consisting of enzymatic assays as they 1 ACS Paragon Plus Environment

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typically have superior selectivity and sensitivity. Enzymatic assays are methods that allow for the measurement of an analyte using a biological component (enzymes, antibodies, etc.) along with a chemical detector and are traditionally used in clinical or point-of-care diagnostics because of their unparalleled sensitivity and selectivity. One example of an enzymatic system in the clinical field is the analysis of an enzyme known as alkaline phosphatase which is a marker for bone growth in young people as well as a marker for bone disease in adults. 20 The second example, and perhaps the most well-known, is the mechanism found within a glucometer where glucose in the user’s blood is consumed by the glucose oxidase enzyme which causes a redox reaction to generate a readable output.21 Additionally, biological sex remained the attribute of interest as it has been wellstudied by our group and provided a solid platform for the development of these single analyte systems. Given our success with the ninhydrin and Bradford chemical assays, it was decided that a single analyte chemical assay would be added to the array of potential methods. One benefit for the development of a chemical assays is that it provides more options for possible amino acid markers as not all amino acids have readily available targeting enzymes. Moreover, chemical assays have an advantage of stability as many chemical components can be left on a shelf or in refrigerator for years at a time, while certain enzymes must be replaced after several weeks or months even if stored in the appropriate conditions. The history of chemical assays specifically for fingerprint analysis is extensive but, at the same time, is rather limited. Of the chemical methods that are used to develop latent fingerprints such as 1,2-diazafluoren-9-one (DFO) which fluoresces when irradiated with blue-green light,22,23 the ninhydrin method – which we have already studied – is the most well-known and widely used. Chemical tests are also fairly well-known in other areas of forensic science as there are chemical field kits that are used for the on-site analysis of drug samples. The most common tests for illicit substances are Marquis, Simon’s and Chen’s test. 24 Aside from forensics, chemical analysis methods are also seen in applications such as water test kits,25 chemical detectors for sensing and determination of different warfare agents,26-29 and the VOCkit system30,31 which is used by the Army for the detection of chemical threat agents such as anthrax, sarin and mustard gas. 30-32 In this manuscript, two systems – one enzymatic assay and one chemical assay – have been developed for the detection and analysis of two individual amino acids. The first system is a threeenzyme cascade for the detection of alanine. This enzymatic assay utilizes alanine transaminase (ALT) which has a specific affinity for alanine, pyruvate oxidase (POx), and horseradish peroxidase (HRP). Here, ALT reacts with alanine in the fingerprint sample and, in turn, produces pyruvate. The pyruvate is then converted to hydrogen peroxide (H2O2) which is then consumed by HRP to oxidize the redox dye which generates a visible blue color, measurable at 405 nm. Previously, the ALT/POx/HRP system has been used as an electrochemical biosensor for assessing injuries.33 The second system is a chemical assay, known as the Sakaguchi test, for the detection of arginine. The Sakaguchi test, created by Schoyo Sakaguchi in 1925, 34 is a relatively sensitive colorimetric assay that specifically detects arginine.35 The specificity of this assay for arginine is due to the presence of the guanidine group in its structure 35 which reacts with α-naphthol and hypobromite under alkaline conditions. This reaction, once complete, generates a visibly distinct red-colored complex that can be spectrophotometrically measured at 500 nm. This wavelength was confirmed by measuring the spectrum of the red-colored complex. Experimental Ethics Statement 2 ACS Paragon Plus Environment


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The Institutional Review Board, Office of Pre-Award and Compliance at the University at Albany have fully approved the experimental protocols demonstrated in this manuscript. These protocols were carried out in accordance to the office’s requirement of obtaining informed consent, in the form of a signature from each volunteer, acknowledging that they are aware of the procedure that will take place, any risks or benefits that may accompany the study, as well as acknowledging that they will not receive any payment for their participation. Informed consent from all volunteers who participated in this research study was obtained. Reagents Used Water used in all of the experiments was ultra-pure (18.2 MΩ·cm) water from PURELAB flex, an ELGA water purification system. EMD hydrochloric acid, manufactured by Fisher Scientific, was used for the amino acid extraction procedure. The following reagents were purchased from Sigma Aldrich: glutamic-pyruvic transaminase from porcine heart (also known as alanine transaminase; ALT, 2.6.1.2), pyruvate oxidase (POx, E.C. 1.2.3.3), horseradish peroxidase Type VI (HRP, 1.11.1.7), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium (ABTS), α-ketoglutaric acid sodium salt (KTG), thiamine pyrophosphate (TPP), flavin adenine dinucleotide disodium salt hydrate (FAD), magnesium chloride (MgCl2), dipotassium phosphate, monopotassium phosphate, α-naphthol, sodium hydroxide (NaOH), ethanol, bromine, L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-asparagine, L-glutamine, L-proline, glycine, Lalanine, L-valine, L-cystine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, β-alanine, L-ornithine, L-lysine, L-tryptophan, L-histidine, L-arginine, and L-citrulline. Instrumentation and Measurements. A Molecular Devices UV/Vis spectrophotometer/plate reader – SpectraMax Plus 38436 containing a Xenon flash lamp – was used to take optical measurements of the samples at λ = 405 nm for the ALT/POx/HRP assay and spectrum measurements from 400 nm to 600 nm for the Sakaguchi test. All measurements were carried out at 37 °C in 96-well microtiter polystyrene plates (PS, Thermo Scientific). Methods for the Detection of Alanine and Arginine The three-enzyme cascade, depicted in Scheme 1, for the analysis of both mimicked and authentic fingerprints was designed and optimized in 0.1 M potassium phosphate buffer pH 7.6 containing 5 U ALT, 1 U POx, 3 U HRP, 1 mM KTG, 5.5 µM FAD, 11 µM TPP, 7 mM MgCl2, and 1 mM ABTS. This cascade system is activated when ALT reacts with the alanine present in the sample. Pyruvate is produced by the interaction of alanine and ALT. It is then converted to H2O2 via POx, which is then consumed by HRP. This results in the Scheme 1. The enzymatic cascade oxidation of ABTS, which is observed spectrophotometrically for recognition of sex via alanine at λ = 405 nm. All components of the assay were added to and content in both mimicked and mixed in the microtiter plate wells prior to the addition of authentic fingerprint samples. alanine to start the reaction. Prior to measurement there was a 4 minute incubation period at 37 °C. Ultimately, the rate of color production (oxidation of ABTS) is proportional to the concentration of alanine present in the sample.

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For the Sakaguchi test, depicted in Scheme 2, several solutions were prepared. First αnaphthol was dissolved in 95% ethanol for a 1.5 mM solution. Second, 160 µL of pure bromine was added to 5% NaOH totaling 25 mL to generate a sodium hypobromite solution. A separate 10% NaOH solution was also needed. For the mimicked samples, 30 µL of both 10% NaOH and α-naphthol were used, along with 5 µL of sodium hypobromite and 150 µL of the mimicked fingerprint sample. However, for the authentic fingerprint samples, 22 µL of 10% NaOH and α-naphthol were used along with 4 µL of sodium hypobromite and 100 µL of the authentic fingerprint sample. The change is volumes were necessary to account for the sample dilution that is seen as a result of the extraction protocol. To begin the reaction process, the appropriate volumes (depending on the sample in use) of 10% NaOH and α-naphthol were added to a 1.5 mL centrifuge tube along with the corresponding volume of either the mimicked or authentic fingerprint sample. The mixture was briefly vortexed and placed in an ice bath for 5 minutes. In the meantime, the 4 µL or 5 µL of Scheme 2. Sakaguchi test for the detection of sodium hypobromite was added to the microtiter arginine from fingerprint content, using both plate wells for authentic fingerprints and mimicked and authentic fingerprint samples. mimicked fingerprints, respectively. Following the incubation period, 175 µL of the incubated mimicked fingerprint sample was added to the wells containing 5 µL of sodium hypobromite and 140 µL of the authentic fingerprint sample was added to the wells containing the 4 µL of sodium hypobromite. Regardless of the sample that was used, all wells were analyzed at 37 °C from 400 nm to 600 nm with a 5 nm step. For validity purposes, both the enzymatic assay and the chemical assay were tested on authentic fingerprints from one male and one female that were taken from 5 different surfaces – a brass door knob, a laminate desktop, a chemical resin lab benchtop, a glass computer screen, and the polyethylene film (PEF) that was also used for the previous experiments. These analyses were performed following the procedures for authentic fingerprint analysis, as described above. Statistical Analysis As in our previous publications, receiver operating characteristic (ROC) analysis was utilized for statistical analysis of the data generated from the mimicked and the authentic fingerprints for both the ALT/POx/HRP assay and the Sakaguchi test. ROC analysis was performed using R-project software37-42 in order to determine the diagnostic potential, of each assay, otherwise known as the ability to correctly differentiate between male and female fingerprints.9-11,17-19 ROC curves and Areas Under the Curve (AUCs) were calculated to estimate the discriminatory power of the respective assays. The AUC of the ROC curve was calculated by the trapezoidal method of integration with the corresponding 95% confidence intervals (CI) as described by De Long et al.42 Using this method, the best threshold (the point at which the signal changes correspond to only one of the groups) that yielded the maximum accuracy was determined. Results To address the potential problem of an enzyme’s varying affinity for all L-amino acids – 4 ACS Paragon Plus Environment


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such as the case of L-amino acid oxidase (LAAO)17 – which could cause an attribute override, a concerted enzymatic assay was developed and Table 1.10 Average Concentrations (mM) of optimized using three enzyme – glutamic-pyruvic Alanine and Arginine for Females and Males transaminase from porcine heart (also known as Derived from Sweat.a. alanine transaminase; ALT, 2.6.1.2), pyruvate oxidase (POx, E.C. 1.2.3.3), and horseradish peroxidase (HRP; E.C. 1.11.1.7). Likewise, the Sakaguchi test was established to address a These values have been previously reported and ninhydrin’s and Bradford’s reliance on multiple were used to prepare mimicked fingerprint samples. analytes. For the ALT/POx/HRP assay, alanine alone was targeted while the Sakaguchi test targeted arginine. It is important to note that the concentrations of these two amino acids – based on previous published data10,17-19 – are at opposite ends of the concentration range. According to this data, alanine is fifth highest concentration and arginine is the lowest concentration. These concentrations can be found in Table 1.10,17-19 Given that this was the first time that biological sex identification was attempted using a single analyte, calibration curves of the expect range of concentrations for each amino acid were generated using the respective assay. In Figure 1, an obvious concentration dependence can be seen from varying concentrations of alanine when analyzed by the ALT/POx/HRP assay. The previously reported average male and female alanine concertation have been used as part of this calibration curve and are denoted by the blue and red stars, respectively. Similarly, Figure 2 represents a calibration curve for the expected range of arginine values was constructed using the Sakaguchi test. The previously reported average male and female arginine values have been denoted in the same manner as in Figure 1.

Figure 1. Calibration curve to demonstrate the dependence of the ALT/POx/HRP assay on the concertation of alanine.

Figure 2. Calibration curve to demonstrate the dependence of the Sakaguchi test on the concertation of arginine.

Once it was determined that both assays would be able to show the difference between a variety of standard concentrations, mimicked sample were created. Previously reported data were utilized to prepare buffer-based solutions that mimicked the levels of all amino acids known to be present in the fingerprints of males and females.10,17-19 The values stated in these statistical studies were not normally distributed, but rather positively skewed and consistent with a log-normal distribution. The parameters of the log-normal distribution were available only for overall amino acid concentrations, while the distribution parameters estimated from the male and female data came from logarithmic untransformed data. The existing parameters for a normal distribution were first modified for a log-normal distribution. For each of the 23 amino acids present in the 5 ACS Paragon Plus Environment

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fingerprint content, random values agreeing with the recalculated parameters of the log–normal distribution in males and females were established using R-project software.37-42 As a result, two sets of concentration values of all 23 amino acids were statistically generated, resulting in 575 different amino acid concentrations for each biological sex which were then arbitrarily grouped together to create 25 samples representing males and 25 samples representing females. The same concentrations and distribution of said concentrations were used for both assays. Following analysis and optimization with mimicked samples, both assays were tested on authentic fingerprint samples. This included both fingerprints from the ideal surface (polyethylene film) as well as those that were taken from the 4 additional surfaces (a brass door knob, a laminate desktop, a chemical resin lab benchtop, a glass computer screen). The same extraction protocol that was used in our previous publications was again used here. 17-,18,19 ALT/POx/HRP Assay for the Detection of Alanine To begin the process of establishing the concept of biological sex identification via a single analyte (amino acid), we established an enzymatic assay. Enzymatic assays are well-known for their specificity for their target analyte and are also relatively sensitive. As previously mentioned, we chose a three-enzyme cascade whose target analyte is only alanine. This was also a beneficial choice since alanine is in the top five most concentrated amino acids in fingerprint content. Figure 3 displays the optical responses for the ALT/POx/HRP assay when used with mimicked fingerprint samples. The output signal is defined as the absorbance of oxidized ABTS as a function of time. As we would anticipate, given that females have higher amino acid concentrations than males, the female responses (red traces) were generally higher than the male responses (blue traces). Unlike what we have seen in the past, there was significant overlap of five male samples with the female samples. Given that the alanine concentrations were statistically generated based on the average value and the fact that this assay only targets one amino acid, this overlap was not unexpected. In the case of the multi-analyte LAAO assay, the response produced is the sum of the response from each amino acid that LAAO has an affinity for. Because of this,

Figure 3. Change in absorbance (ΔAbs.) at 405 nm corresponding to the oxidization of ABTS upon the reaction of the ALT/POx/HRP system with mimicked fingerprint samples. There was a 4 min. incubation at 37 °C prior to measurement. The bottom red traces indicate the female samples and the top blue traces indicate the male samples.

Figure 4. Trade-off between sensitivity and specificity is shown as an ROC curve with an AUC of 82%. This is the probability for the presented assay to correctly distinguish between males and females based on the amino acid’s concentrations. The optimum cut off point was chosen with a sensitivity of 80% and specificity of 100%. Random choice is denoted by the gray diagonal line.



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there is a high probability that some amino acids would result in a high absorbance while other result in a low absorbance, and thus, the actual response is somewhere in the middle. The use of a single analyte system almost completely removes this potential interference, and therefore you are seeing a direct result of the analyte. We performed ROC analysis on this data and the best absorbance threshold of 0.073 was determined. This value balanced the trade-off that occurs between sensitivity and specificity and is the most precise cutoff point for the differentiation between male and female fingerprint samples. The area under the ROC curve, also known as AUC, was approximated via the trapezoidal integration method, and the corresponding 95% confidence interval (CI) was estimated.42 The AUC was estimated at 0.816 (95% CI, 0.6675-0.9645) from the ROC curve (Fig. 4), which means that the ALT/POx/HRP enzymatic assay has an 82% probability of correctly differentiating between male and female mimicked fingerprints. Despite this being one of the lowest ROC values to date, we decided to evaluate whether or not this assay was viable for authentic fingerprints as well as determine whether or not the use of authentic fingerprints would improve the ROC. Because the amino acid concentrations in the model solutions were chosen to follow the published distributions that are relevant for males and females, authentic applications were expected to generate signals that mirror the signal distribution of the mimicked samples. These fingerprints were collected from two groups of volunteers, males and females, on the PEF according to an established procedure described by Croxton11 and extracted according to the procedure in our previous publications.17-19 As expected, based on previously published results, 17-19 the aqueous acidic solution collected from the fingerprint extraction consisted of the amino acid – alanine – needed for this analysis, while the nonpolar material stayed on the PEF. As can be seen in Figure 5, there is much less overlap between female and male samples as compared to the mimicked samples. ROC analysis was subsequently performed and the best absorbance threshold of 0.9984 was identified. The AUC was estimated at 0.998 (95% CI, 0.994-1.0) from the ROC curve (Fig. 6), which means that the ALT/POx/HRP enzymatic assay has a 99.8% probability of correctly differentiating

Figure 5. Change in absorbance (ΔAbs.) at 405 nm corresponding to the oxidization of ABTS upon the reaction of the ALT/POx/HRP system with authentic fingerprint samples. There was a 4 min. incubation at 37 °C prior to measurement. The bottom red traces indicate the female samples and the top blue traces indicate the male samples.

Figure 6. Trade-off between sensitivity and specificity is shown as an ROC curve with an AUC of 99.8%. This is the probability for the presented assay to correctly distinguish between males and females based on the amino acid’s concentrations. The optimum cut off point was chosen with a sensitivity of 100% and specificity of 96%. Random choice is denoted by the gray diagonal line.


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between authentic male and female fingerprints. In spite of the fact that we used the appropriate procedure 37-42 to generate the concentrations for the mimicked samples in order to stay close to the original distribution drawn from a population based on a large data set that provided real-life context, the results shown here are simply an aproximation of it. The idea is to mimic complicated real-life data in a simpler form – to have concentrations available for practical use in experiments. The actual samples will always vary, to some extent, compared to the mimicked samples simply because mimicked samples cannot capture all factors of the actual samples in addition to the fact that actual samples are drawn from a different population, often smaller, than the mimicked samples. Sakaguchi Test for the Detection of Arginine As previously mentioned, the Sakaguchi test was developed in 1925 and is reliant on the presence of the guanidine group within the structure of arginine as well as an alkaline environment. As with the enzymatic assay, mimicked fingerprint samples were analyzed first. Unlike the ALT/POx/HRP enzymatic assay, which involved the oxidation of the dye over time, the Sakaguchi test was much more instantaneous. As a result, kinetic analysis was unable to capture the reaction because the color change had occurred almost immediately. Because of this, we measured the absorbance at the end of the reaction across a spectrum of wavelengths from 400 nm to 600 nm, with a maximum wavelength at 500 nm. Figure 7 depicts the optical response of the Sakaguchi test when analyzing mimicked fingerprint samples. As anticipated, the female samples (red) generated high absorbance values in comparison to the male samples (blue). Despite having such low concentrations of arginine, the response of the Sakaguchi test generated impressively high absorbance values. ROC analysis was performed just as with the ALT/POx/HRP assay. The best absorbance threshold of 0.130 was identified and the AUC was estimated at 1.0 (95% CI, 1.0-1.0) from the ROC curve (Fig. 8), which means that the Sakaguchi test has a 100% probability of correctly differentiating between male and female mimicked fingerprints.

Figure 7. Absorbance of red-colored complex generated from the Sakaguchi test with mimicked fingerprint samples from 400 nm to 600 nm with a 5 nm step. The maximum absorbance was identified as 500 nm. The red traces correspond to the mimicked female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples.




Figure 9. Absorbance of red-colored complex generated from the Sakaguchi test with mimicked fingerprint samples from 400 nm to 600 nm with a 5 nm step. The maximum absorbance was identified as 500 nm. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples

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The impressive response of the Sakaguchi test with mimicked fingerprints gave us confidence that it would be successful with authentic fingerprints. Again, 25 authentic female fingerprints and 25 authentic male fingerprints were analyzed via the Sakaguchi test. As described in the Methods for the Detection of Alanine and Arginine section, the protocol for the analysis of authentic fingerprint samples was slightly modified. Figure 9 displays the optical readout for the authentic fingerprint samples. An impressive surprise when looking at these results was that despite there being several steps that could potentially decrease the amount of arginine present in the sample, such as the transfer to the PEF and the dilution during the extraction, the absorbance values have decreased only slightly. For statistical comparison, ROC analysis was performed. For this experiment, the best absorbance threshold was identified as 0.079 and the AUC was estimated at 1.0 (95% CI, 1.0-1.0) from the ROC curve (Fig. 10). Once again, this means that the Sakaguchi test assay has a 100% probability of correctly differentiating between authentic male and female fingerprints. Evaluation of Surfaces To conclude this study, just as in our previous work, it was important to bring these assays full-circle in a forensic context. This meant that it was necessary to ensure the validity of both assays in real crime scene scenarios. The extraction protocol and both the ALT/POx/HRP assay and the Sakaguchi test were further tested on fingerprints from various surfaces that could be found at a crime scene. Five authentic female fingerprints and five authentic male fingerprints were deposited onto four surfaces including a brass door knob, a laminate desktop, a chemical resin lab benchtop, a glass computer screen. The PEF was also used as the fifth surface and thus was the control. Again, this totaled 25 female fingerprints and 25 male fingerprints. The PEF was also used to remove the fingerprints from the respective surfaces. Figure 11 and 12 demonstrate that the extraction protocol along with the ALT/POx/HRP assay and the Sakaguchi test, respectively, are capable of identifying a female fingerprint versus a male fingerprint, regardless of the surface from 9 ACS Paragon Plus Environment

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which it was taken. Figure 11 is representative of the average (n=5) response of the five samples for each surface and each sex at 405 nm after 600 seconds using the ALT/POx/HRP assay. Just as in the previous experiments, a 4 minute incubation time at 37 °C was required. It is important to note that male fingerprints from the four test surfaces were not included in the graph as they did not generate any signal. This was expected given that there was a significant decrease in an already fairly low absorbance when comparing the female fingerprints from the PEF to the female fingerprints from the test surfaces. The inability to see male fingerprints from surfaces was also seen in our first fingerprint publication.17 Despite this significant difference between female fingerprints on the control surface and the female fingerprints on the other surfaces, it was still possible to identify a female fingerprint from a male fingerprint as the absorbance remained higher than the response from the ideal male fingerprint samples. On the other hand, Figure 12 represents the average (n=5) response of the five sample for each surface and each sex at 500 nm using the Sakaguchi test. The protocol for this experiment followed the procedure used for the analysis of authentic fingerprints, as described above. Here male and female fingerprints were able to be seen from all five surfaces. This was anticipated since the starting absorbance was fairly high. As can be seen, even with a significant decrease in absorbance when going from the ideal surface to the test surfaces, female fingerprints could still be distinguished from male fingerprints. Error bars were included on both bar graphs to demonstrate the success of the fingerprint transfer from the surfaces to the PEF and extraction of the amino acid content as well as to show that the error is low enough that is does not play a role in differentiating between a male and female fingerprint.

Figure 11. Bar diagram of the average change in absorbance (n=5) for each surface and each sex at 405 nm after 600 seconds. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples. Error bars are included in the figure to demonstrate the efficacy of transferring the fingerprint from the respective surface to the PEF and subsequently performing the extraction. The abbreviation PEF represents the polyethylene film and Comp represents a computer screen.

Figure 12. Bar diagram of the average absorbance (n=5) for each surface and each sex at 500 nm. The red traces correspond to the authentic female fingerprint samples and the blue traces correspond to the authentic male fingerprint samples. Error bars are included in the figure to demonstrate the efficacy of transferring the fingerprint from the respective surface to the PEF and subsequently performing the extraction. The abbreviation PEF represents the polyethylene film and Comp represents a computer screen.

Conclusion Based on all of the data presented here, the hypothesis has been confirmed that a single 10 ACS Paragon Plus Environment


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amino acid – regardless of concentration – can be correlated to biological sex. This also indicates that any amino acid can potentially be used to do so. However, even though biological sex has now been well-studied, this work indicates that with the proper investigation and data, it is entirely possible to find a single biological marker – amino acid or other metabolite – that can be directly correlated to a single originator attribute other than biological sex. This research concept is currently under development in our lab. The work shown also demonstrates that a chemical assay may be a more viable option for further studies. Perhaps the most important aspect to note is that the amino acids that were analyzed in this project cover the vast range of concentrations known to be in fingerprint content. Alanine is has the fifth highest concentration and arginine has the lowest concentration. Even with arginine having the lowest concentration, the Sakaguchi test generated a larger separation between male and female responses in comparison to the ALT/POx/HRP assay. Despite that the ALT/POx/HRP cascade did not necessarily perform as well as the Sakaguchi test, it is important to acknowledge both. This is because the ultimate goal is to have multiple systems available for all of the different amino acids and it is possible that enzymatic assays may be beneficial for amino acids with higher concentrations and chemical assays may be more beneficial for amino acids with lower concentrations. The two single analyte systems described above for the purpose of distinguishing between fingerprint samples obtained from both males and females have proven to be reliable and reproducible. The ROC analysis conducted using 50 mimicked fingerprint samples and the ALT/POx/HRP system generated statistics supporting that it is possible to determine the sex of the fingerprint originator. These results concluded that there was an 82% chance of correctly determining the sex of the fingerprint originator. Authentic fingerprints were subsequently analyzed using a reliable sample extraction protocol to remove the necessary substrate – alanine. The ROC analysis of the data from the authentic fingerprint samples further demonstrated the ability of the enzymatic assay to differentiate between authentic male and female fingerprint samples as it reported a 99.8% chance of correctly identifying the biological sex of the originator. Additionally, a chemical assay – the Sakaguchi test –for the detection of arginine was optimized for the ability to identify biological sex from fingerprints. Mimicked samples were analyzed first and ROC analysis determined that this test had a 100% chance of correctly discerning a female fingerprint from a male fingerprint. Using this as motivation, authentic fingerprints were analyzed next and again the ROC analysis determined that the assay had a 100% chance of correctly identifying the biological sex of the fingerprint originator. Surface experiments concluded this project. The purpose of these experiments were to ensure that both assays could still perform successfully even when the fingerprints were from nonideal surfaces so as to uphold real crime scene scenarios as much as possible. In the case of the ALT/POx/HRP assay, male fingerprints from the test surfaces were unable to generate a significant signal. Contributing factors for this observation include the significant loss and dilution of the analyte that occurs during the transfer from the surface to the PEF and the extraction process. This is evident by the differences among the female fingerprints alone. However, the male fingerprints from the ideal surface were still lower than the female fingerprints from the test surfaces, allowing for differentiation. As for the Sakaguchi test, all male fingerprints were able to generate absorbance results and all were lower than the female samples. To summarize, both assays were able to identify the biological sex of the fingerprint originator regardless of the surface. Ultimately, the results displayed here are the first of their kind, in that the single amino acid assays – one that has a specific affinity for alanine and one that has a specific affinity for arginine – are fully capable of distinguishing between sexes from the contents of fingerprints. The 11 ACS Paragon Plus Environment

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ability to successfully establish single analyte systems for the identification of originator attributes opens the door for the potential correlation of one analyte to one originator attribute. Furthermore, these findings demonstrate the possibility of creating a platform that is capable of identifying multiple originator attributes from a single fingerprint in order to establish a profile of a person of interest.



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