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.

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.

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.

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.

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.

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.

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.

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.

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 model system for targeted drug release triggered by biomolecular signals logically processed through enzyme logic networks.

A new Sense-and-Act system was realized by the integration of a biocomputing system, performing analytical processes, with a signal-responsive electrode. A drug-mimicking release process was triggered by biomolecular signals processed by different logic networks, including three concatenated AND logic gates or a 3-input OR logic gate. Biocatalytically produced NADH, controlled by various combinations of input signals, was used to activate the electrochemical system. A biocatalytic electrode associated with signal-processing "biocomputing" systems was electrically connected to another electrode coated with a polymer film, which was dissolved upon the formation of negative potential releasing entrapped drug-mimicking species, an enzyme-antibody conjugate, operating as a model for targeted immune-delivery and consequent "prodrug" activation. The system offers great versatility for future applications in controlled drug release and personalized medicine.

Biocatalytic analysis of biomarkers for forensic identification of gender.

A new biocatalytic assay analyzing the simultaneous presence of creatine kinase (CK) and alanine transaminase (ALT) was developed aiming at the recognition of biofluids of different gender for forensic applications. Knowing the difference in the concentrations of CK and ALT enzymes in the blood of healthy adults of male and female groups we mimicked the samples of different gender with various CK-ALT concentrations. The analysis was performed using a multi-enzyme/multi-step biocatalytic cascade where the differences in both included enzymes resulted in an amplified difference in the final analytical response. The analysis performed in human serum solutions allowed discrimination of samples corresponding to male/female groups. The robustness of the developed analysis allowed determination of the gender for serum solutions after their drying and ageing at least for 1 hour. Importantly for forensic applications, reaction with a chromogenic reactant nitroblue tetrazolium allowed qualitative discrimination of the "male" and "female" samples by the naked eye.

An enzyme-based reversible CNOT logic gate realized in a flow system.

An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD(+) and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomolecular realization of a CNOT gate is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.

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.

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.

Evaluation of Fentanyl Exposure Effects on Butyrylcholinesterase Activity as a Tool for Future On-Site Detection Methods.

The prominence of fentanyl and fentanyl analogues or Fentanyl Related Substances (FRS) has driven a nationwide crisis of opioid overdoses, which significantly presents an issue for public health and safety. Originally developed for medical purposes, fentanyl and FRS have become critical contributors to opioid overdose deaths due to their distribution, availability, and potency. This study examined toxicodynamic properties between butyrylcholinesterase (BChE) and fentanyl analogues via Ellman's assay. The enzymatic function of BChE was significantly inhibited by each of the 5 fentanyl analogues tested, which indicates the potential for utilization of this interaction. This reaction can be immobilized for a portable, single-use kit to detect FRS directly from any surface on-site. This would immensely benefit society by reducing the frequency of exposure and overdoses by providing additional safety measures to law enforcement and first responders.

Biocatalytic analysis of biomarkers for forensic identification of ethnicity between Caucasian and African American groups.

A new biocatalytic assay analyzing the simultaneous presence of creatine kinase (CK) and lactate dehydrogenase (LDH) was developed aiming at the recognition of biofluids of different ethnic origins for forensic applications. Knowing the difference in the concentrations of CK and LDH in the blood of healthy adults of two ethnical groups, Caucasian (CA) and African American (AA), and taking into account the distribution pattern, we mimicked the samples of different ethnic origins with various CK-LDH concentrations. The analysis was performed using a multi-enzyme/multi-step biocatalytic cascade where the differences in both included enzymes resulted in an amplified difference in the final analytical response. The statistically established analytical results confirmed excellent probability to distinguish samples of different ethnic origins (CA vs. AA). The standard enzymatic assay routinely used in hospitals for the analysis of CK, performed for comparison, was not able to distinguish the difference in samples mimicking blood of different ethnic origins. The robustness of the proposed assay was successfully tested on dried/aged serum samples (up to 24 h) - in order to mimic real forensic situations. The results obtained on the model solutions were confirmed by the analysis of real serum samples collected from human subjects of different ethnic origins.

Electrode interfaces switchable by physical and chemical signals for biosensing, biofuel, and biocomputing applications.

This review outlines advances in designing modified electrodes with switchable properties controlled by various physical and chemical signals. Irradiation of the modified electrode surfaces with various light signals, changing the temperature of the electrolyte solution, application of a magnetic field or electrical potentials, changing the pH of the solutions, and addition of chemical/biochemical substrates were used to change reversibly the electrode activity. The increasing complexity in the signal processing was achieved by integration of the switchable electrode interfaces with biomolecular information processing systems mimicking Boolean logic operations, thus allowing activation and inhibition of electrochemical processes on demand by complex combinations of biochemical signals. The systems reviewed range from simple chemical compositions to complex mixtures modeling biological fluids, where the signal substrates were added at normal physiological and elevated pathological concentrations. The switchable electrode interfaces are considered for future biomedical applications where the electrode properties will be modulated by the biomarker concentrations reflecting physiological conditions.