Who Shot the Bullet? Projectile Composition Characterization as an Evolutionary Method for Enhancement of Ballistics Evidence Analysis.

Toolmark and Firearm examiners' opinions have fallen under scrutiny as inadmissible ballistics evidence has led to the possibility of wrongful convictions and cold cases that could have been solved with the presence of a physical bullet, casing, and/or weapon at the crime scene. This research provides a solution for subjective-based conclusions and the absence of physical evidence altogether. Analysis of bullet material using Atomic Absorption Spectroscopy (AAS) has distinguished bullet composition between manufacturers from a surface scratch. This provides proof of concept that, when a bullet strikes a surface, metal deposits can be extracted and analyzed to corroborate microscopy techniques that currently violate Daubert criteria. Further studies could also provide results to distinguish barrel manufacturers from fired bullets and casings. This novel method of analysis can pave the way for crime scene collection procedures in the absence of physical evidence and provide an increase in scientific value to the expert's conclusions.

Metabolite monitoring concept for the biometric identification of individuals from the skin surface.

This study aims at proof of concept that constant monitoring of the concentrations of metabolites in three individuals' sweat over time can differentiate one from another at any given time, providing investigators and analysts with increased ability and means to individualize this bountiful biological sample. A technique was developed to collect and extract authentic sweat samples from three female volunteers for the analysis of lactate, urea, and L-alanine levels. These samples were collected 21 times over a 40-day period and quantified using a series of bioaffinity-based enzymatic assays with UV-vis spectrophotometric detection. Sweat samples were simultaneously dried, derivatized, and analyzed by a GC-MS technique for comparison. Both UV-vis and GC-MS analysis methods provided a statistically significant MANOVA result, demonstrating that the sum of the three metabolites could differentiate each individual at any given day of the time interval. Expanding upon previous studies, this experiment aims to establish a method of metabolite monitoring as opposed to single-point analyses for application to biometric identification from the skin surface.

Implantable bioelectrochemistry: 70 years across "Cyborg" organisms and logically operated bioelectronics.

Professor Evgeny Katz (Department of Chemistry and Biomolecular Science, Clarkson University, USA) was born on 11th August 1952, and he turned 70 years old last summer. This special collection entitled Implanted Enzymatic Fuel Cells and Biosensors: Fundamentals to Applications is dedicated to Evgeny on this landmark occasion. This brief preface gives some personal insights into Evgeny's career beyond the scientific perspective.

Determination of Time since Deposition of Fingerprints via Colorimetric Assays.

Past investigations involving fingerprints have revolved heavily around the image of the fingerprint-including the minutiae, scarring, and other distinguishing features-to visually find a match to its originator. Recently, it has been proven that the biochemical composition can be used to determine originator attributes, such as sex, via chemical and enzymatic cascades. While this provides pertinent information about the originator's identity, it is not the only piece of information that can be provided. This research was designed with three goals in mind: (1) identify how long it would take before an aged female fingerprint could no longer be differentiated from a male fingerprint, (2) identify a correlation between the data collected and a specific time since deposition (TSD) time point, and (3) identify if a specific amino acid could be contributing to the decreasing response seen for the aging fingerprints. Using ultraviolet-visible (UV-vis) spectroscopy, aged fingerprints were evaluated over the course of 12 weeks via three chemical assays previously used for fingerprint analysis-the ninhydrin assay, the Bradford assay, and the Sakaguchi assay. As fingerprints age, the conditions they are exposed to cause the biochemical composition to decompose. As this occurs, there is less available to be detected by analytical means. This results in a less intense color production and, thus, a lower measured absorbance. The results displayed here afforded the ability to conclude that all three goals set forth for this research were accomplished-a female fingerprint can be differentiated from a male fingerprint for at least 12 weeks, UV-vis data collected from aged fingerprints can be correlated to a TSD range but not necessarily a specific time point, and the decomposition of at least a single amino acid can afford the ability to estimate the TSD of the fingerprint.

Recent Advances in Noninvasive Biosensors for Forensics, Biometrics, and Cybersecurity.

Recently, biosensors have been used in an increasing number of different fields and disciplines due to their wide applicability, reproducibility, and selectivity. Three large disciplines in which this has become relevant has been the forensic, biometric, and cybersecurity fields. The call for novel noninvasive biosensors for these three applications has been a focus of research in these fields. Recent advances in these three areas has relied on the use of biosensors based on primarily colorimetric assays based on bioaffinity interactions utilizing enzymatic assays. In forensics, the use of different bodily fluids for metabolite analysis provides an alternative to the use of DNA to avoid the backlog that is currently the main issue with DNA analysis by providing worthwhile information about the originator. In biometrics, the use of sweat-based systems for user authentication has been developed as a proof-of-concept design utilizing the levels of different metabolites found in sweat. Lastly, biosensor assays have been developed as a proof-of-concept for combination with cybersecurity, primarily cryptography, for the encryption and protection of data and messages.

Step toward Roadside Sensing: Noninvasive Detection of a THC Metabolite from the Sweat Content of Fingerprints.

The sudden increase in states legalizing marijuana has forced law enforcement into a situation where the use and consumption are legal, but there are no limitations for what is acceptable for driving or operating machinery. Using ultraviolet-visible (UV-vis) spectroscopy, fingerprints from volunteers who had used marijuana were analyzed via a competitive immunoassay for the detection of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component of marijuana, and 11-nor-9-carboxy-THC (THC-COOH), one of the main metabolites produced in the body following the use/consumption of THC-related products. In this research, the THC-COOH metabolite and the enzyme-labeled conjugate compete against each other as the antigens for the system. The antibody used in this assay has a greater affinity for the metabolite; so, as its concentration increases, the absorbance of the system decreases due to less binding of the enzyme-labeled conjugate.

Noninvasive Concept for Optical Ethanol Sensing on the Skin Surface with Camera-Based Quantification.

Law enforcement and the general public do not yet have adequate means of assessing and preventing drunk driving. Blood alcohol concentration (BAC) is unable to be determined on-site, as it typically requires the use of complex chromatographic methods. Breathalyzers have been well established in law enforcement for correlating breath alcohol concentrations (BrAC) to BAC estimations, as they involve portable equipment with rapid analysis times. Although these BrAC measurements allow police officers to determine probable cause and to arrest an intoxicated driver at the scene, the results are preliminary and are not often considered as evidence in court. A new, noninvasive method was developed to assess an individual's level of intoxication based on the presence of ethanol in sweat on the skin surface. This intuitive system uses two enzymes, alcohol oxidase and horseradish peroxidase, to correlate ethanol sweat concentrations to the production of a color that is visible to the naked eye. The results of the controlled drinking study demonstrate the ability of both the spectrophotometric and the visualization system to quantify the amount of ethanol within authentic sweat samples collected from individuals who had consumed an alcoholic beverage. The pictorial analysis allows for the system to be analyzed without the use of a UV-vis spectrophotometer. With this method, a smartphone application would be capable of documenting and evaluating the intoxication levels of an individual based on sweat ethanol levels. The developed alcohol sensing system has the potential to impact both the general public and law enforcement, as well as the fields of forensic and biomedical science.

Symmetric-Key Encryption Based on Bioaffinity Interactions.

The research presented here shows a bridge between biochemistry and cryptography. Enzyme-based assays were used in a new methodology linked to ciphers and cipher systems. Three separate enzyme assays, alkaline phosphatase (ALP) (E.C. 3.1.3.1), lysozyme (E.C. 3.2.1.17), and horseradish peroxidase (HRP) (E.C. 1.11.1.7), were used to create a cipher key in order to encrypt a message. By choosing certain parameters for one's experiment that are performed in the same way as a person receiving the message, correct encryption and decryption keys would be produced, resulting in a correct encryption and decryption of a message. It is imperative that both parties perform the same experiment under the same conditions in order to correctly interpret the message. Bioaffinity-based assays, in particular enzymatic assays, provide a specific, yet flexible mechanism to use for the encryption of messages. Because of the nature of this process there are a multitude of sets of parameters that may be chosen, each of which would result in a different key being produced, heightening the security and the robustness of the method. This paper shows that by using this concept of forming encryption keys using a bioaffinity-based approach, one is able to properly encrypt and decrypt a message, which could be viable for other biochemically based techniques.

Fluorescence of 1,2-Indanedione with Amino Acids Present in the Fingerprint Residue: Application in Gender Determination.

1,2-indanedione is used for latent fingerprint visualization on porous surfaces. In this paper, fluorescence spectra of 1,2-indanedione after reacting with 21 individual amino acids present in latent fingerprints residue were measured in water-methanol solutions. The fluorescence intensity depends on the amino acid used, while the fluorescence peak does not change much. The concentration of amino acids in fingerprint residue in females is almost the double of their concentration in males. This property combined with fluorescence of 1,2-indanedione-amino acids compounds is used for gender determination, by comparing the fluorescence intensity peaks in the same experimental conditions. In this preliminary study, the fluorescence signal from the samples representing females was almost two times the signal for samples representing males. In addition to the reduction by almost 50% of the number of suspects in a criminal research case, these results could be helpful in gaining some knowledge about the papillary residue composition.

Metabolite Biometrics for the Differentiation of Individuals.

Sweat is a biological fluid present on the skin surface of every individual and is known to contain amino acids as well as other low molecular weight compounds. (1) Each individual is inherently different from one another based on certain factors including, but not limited to, his/her genetic makeup, environment, and lifestyle. As such, the biochemical composition of each person greatly differs. The concentrations of the biochemical content within an individual's sweat are largely controlled by metabolic processes within the body that fluctuate regularly based on attributes such as age, sex, and activity level. Therefore, the concentrations of these sweat components are person-specific and can be exploited, as presented here, to differentiate individuals based on trace amounts of sweat. For this concept, we analyzed three model compounds-lactate, urea, and glutamate. The average absorbance change from each compound in sweat was determined using three separate bioaffinity-based systems: lactate oxidase coupled with horseradish peroxidase (LOx-HRP), urease coupled with glutamate dehydrogenase (UR-GlDH), and glutamate dehydrogenase alone (GlDH). After optimization of a linear dependence for each assay to its respective analyte, analysis was performed on 50 mimicked sweat samples. Additionally, a collection and extraction method was developed and optimized by our group to evaluate authentic sweat samples from the skin surface of 25 individuals. A multivariate analysis of variance (MANOVA) test was performed to demonstrate that these three single-analyte enzymatic assays were effectively used to identify each person in both sample sets. This novel sweat analysis approach is capable of differentiating individuals, without the use of DNA, based on the collective responses from the chosen metabolic compounds in sweat. Applications for this newly developed, noninvasive analysis can include the field of forensic science in order to differentiate between individuals as well as the fields of homeland security and cybersecurity for personal authentication via unlocking mechanisms in smart devices that monitor metabolites. Through further development and analysis, this concept also has the potential to be clinically applicable in monitoring the health of individuals based on particular biomarker combinations.

Fingerprint Analysis: Moving Toward Multiattribute Determination via Individual Markers.

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 has been 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 has a significantly higher concentration than arginine in the fingerprint content of both males and females. 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.

Promises and Challenges in Continuous Tracking Utilizing Amino Acids in Skin Secretions for Active Multi-Factor Biometric Authentication for Cybersecurity.

We consider a new concept of biometric-based cybersecurity systems for active authentication by continuous tracking, which utilizes biochemical processing of metabolites present in skin secretions. Skin secretions contain a large number of metabolites and small molecules that can be targeted for analysis. Here we argue that amino acids found in sweat can be exploited for the establishment of an amino acid profile capable of identifying an individual user of a mobile or wearable device. Individual and combinations of amino acids processed by biocatalytic cascades yield physical (optical or electronic) signals, providing a time-series of several outputs that, in their entirety, should suffice to authenticate a specific user based on standard statistical criteria. Initial results, motivated by biometrics, indicate that single amino acid levels can provide analog signals that vary according to the individual donor, albeit with limited resolution versus noise. However, some such assays offer digital separation (into well-defined ranges of values) according to groups such as age, biological sex, race, and physiological state of the individual. Multi-input biocatalytic cascades that handle several amino acid signals to yield a single digital-type output, as well as continuous-tracking time-series data rather than a single-instance sample, should enable active authentication at the level of an individual.

Bioaffinity-based assay for the sensitive detection and discrimination of sweat aimed at forensic applications.

Sweat is a well-known piece of biological evidence that is actually used much less than expected. Biological samples are important because their components can often provide some type of information about a person-of-interest. Sweat, in particular, is important because of its DNA content which can be extracted and analyzed to provide information that can be imperative to a criminal investigation. While it is a very important source of forensic information, the methods for detection and discrimination of sweat are limited, causing it to be overlooked during evidence collection. This manuscript presents a biocatalytic method for sweat detection that utilizes an enzyme cascade system that has the capability to detect trace amounts of sweat and distinguish it from saliva, even after the sample has dried. The results show the initial calibration studies performed to insure that the cascade performs well using both mimicked and authentic sweat samples which have components that could negatively affect the enzymes needed for the analysis. The method presented here also has the potential to be adapted for on-site analysis. The initial results of the development of a sweat-sensitive strip are shown here.

Coomassie Brilliant Blue G-250 Dye: An Application for Forensic Fingerprint Analysis.

The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins. However, the ability of the dye to react with a small group of amino acids (arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan) makes it a viable chemical assay for fingerprint analysis in order to identify the biological sex of the fingerprint originator. It is recognized that the identification of biological sex has been readily accomplished using two other methods; however, both of those systems are reliant upon a large group of amino acids, 23 to be precise. The Bradford assay, described here, was developed specifically to aid in the transition from targeting large groups of amino acids, as demonstrated in the previous studies, to targeting only a single amino acid without compromising the intensity of the response and/or the ability to differentiate between two attributes. In this work, we aim to differentiate between female fingerprints and male fingerprints.

Ages at a Crime Scene: Simultaneous Estimation of the Time since Deposition and Age of Its Originator.

Blood is a major contributor of evidence in investigations involving violent crimes because of the unique composition of proteins and low molecular weight compounds present in the circulatory system, which often serve as biomarkers in clinical diagnostics. It was recently shown that biomarkers present in blood can also identify characteristics of the originator, such as ethnicity and biological sex. A biocatalytic assay for on-site forensic investigations was developed to simultaneously identify the age range of the blood sample originator and the time since deposition (TSD) of the blood spot. For these two characteristics to be identified, the levels of alkaline phosphatase (ALP), a marker commonly used in clinical diagnostics corresponding to old and young originators, were monitored after deposition for up to 48 h to mimic a crime scene setting. ALP was chosen as the biomarker due to its age-dependent nature. The biocatalytic assay was used to determine the age range of the originator using human serum samples. By means of statistical tools for evaluation and the physiological levels of ALP in healthy people, the applicability of this assay in forensic science was shown for the simultaneous determination of the age of the originator and the TSD of the blood spot. The stability of ALP in serum allows for the differentiation between old and young originators up to 2 days after the sample was left under mimicked crime scene conditions.

New Horizons for Ninhydrin: Colorimetric Determination of Gender from Fingerprints.

In the past century, forensic investigators have universally accepted fingerprinting as a reliable identification method via pictorial comparison. One of the most traditional detection methods uses ninhydrin, a chemical that reacts with amino acids in the fingerprint content to produce the blue-purple color known as Ruhemann's purple. It has recently been demonstrated that the amino acid content in fingerprints can be used to differentiate between male and female fingerprints. Here, we present a modified approach to the traditional ninhydrin method. This new approach for using ninhydrin is combined with an optimized extraction protocol and the concept of determining gender from fingerprints. In doing so, we are able to focus on the biochemical material rather than exclusively the physical image.

Forensic Identification of Gender from Fingerprints.

In the past century, forensic investigators have universally accepted fingerprinting as a reliable identification method, which relies mainly on pictorial comparisons. Despite developments to software systems in order to increase the probability and speed of identification, there has been limited success in the efforts that have been made to move away from the discipline's absolute dependence on the existence of a prerecorded matching fingerprint. Here, we have revealed that an information-rich latent fingerprint has not been used to its full potential. In our approach, the content present in the sweat left behind-namely the amino acids-can be used to determine physical such as gender of the originator. As a result, we were able to focus on the biochemical content in the fingerprint using a biocatalytic assay, coupled with a specially designed extraction protocol, for determining gender rather than focusing solely on the physical image.

Forensic determination of blood sample age using a bioaffinity-based assay.

A bioaffinity-driven cascade assay was developed to determine the time elapsed from the point a blood sample was left at a crime scene to the point of discovery. Two blood markers, creatine kinase (CK) and alanine transaminase (ALT), were utilized to determine the age of the blood spot based on their natural denaturation processes. The analysis with the proposed bioassay was performed in human serum samples, which underwent the aging process under environmental conditions that could be expected at crime scenes. The concentration of the markers in the sample was based on physiological levels present in healthy adults. These two markers were concerted in a biocatalytic cascade composed of two parallel subsystems, with each of them following the activity of one marker. Both markers have very distinct denaturation rates which would not allow them to be used in a single marker setup while still providing satisfactory results. However, by parallel tunable monitoring of both markers, it is possible to provide information of the blood sample age with low temporal error for a prolonged period of time. To mimic a possible real crime scene situation ­ the reliability of the proposed assay was then successfully tested on dried/aged serum samples (up to 5 days old) in environments with different temperatures.

A biocatalytic cascade with several output signals--towards biosensors with different levels of confidence.

The biocatalytic cascade based on enzyme-catalyzed reactions activated by several biomolecular input signals and producing output signal after each reaction step was developed as an example of a logically reversible information processing system. The model system was designed to mimic the operation of concatenated AND logic gates with optically readable output signals generated at each step of the logic operation. Implications include concurrent bioanalyses and data interpretation for medical diagnostics.