Combining surface-enhanced Raman spectroscopy (SERS) and paper spray mass spectrometry (PS-MS) for illicit drug detection.

There is an ongoing effort in the US illicit drug market to make new psychoactive compounds more potent and addictive. Due to continuous chemical modifications, many fentanyl analogs are developed and mixed with more traditional illicit drugs, such as cocaine and heroin. Detecting fentanyl and fentanyl analogs in these illicit drug mixtures has become more crucial because of the increased potency and associated health risks. Most confirmatory procedures require time-consuming and expensive, highly sophisticated laboratory equipment and experimental procedures, which can delay critical information that might save a victim or find a suspect. In this study, we propose miniaturizing and accelerating this process by combining surface-enhanced Raman spectroscopy (SERS) analysis and paper spray mass spectrometry (PS-MS). For this aim, dual-purposed paper substrates were developed through soaking in Au/Ag nanostars suspensions. These novel, in-house prepared paper SERS substrates showed stability for up to four weeks with and without the presence of drug compounds. Fentanyl analogs with similar SERS spectra were differentiated by coupling with PS-MS. The limit of detection (LOD) for fentanyl on the paper substrates is 34 µg/mL and 0.32 µg/mL for SERS and PS-MS, respectively. Fentanyl and fentanyl analogs show selective SERS enhancement that helped to detect trace amounts of these opioids in heroin and cocaine street samples. In short, we propose the combination of SERS/PS-MS by using modified paper substrates to develop cost-effective, sensitive, rapid, portable, reliable, and reproducible methods to detect illicit drugs, especially trace amounts of fentanyl and fentanyl analogs in illicit drug mixtures. The combination of these two category A techniques allows for the identification of illicit drugs according to the SWGDRUG guidelines.

Differentiating Structurally Similar Fentanyl Analogs by Comparing Density Functional Theory (DFT) Calculations and Surface-Enhanced Raman Spectroscopy (SERS) Results.

Fentanyl and fentanyl analogs are the main cause of recent overdose deaths in the United States. The presence of fentanyl analogs in illicit drugs makes it difficult to estimate their potencies. This makes the detection and differentiation of fentanyl analogs critically significant. Surface-enhanced Raman spectroscopy (SERS) can differentiate structurally similar fentanyl analogs by yielding spectroscopic fingerprints for the detected molecules. In previous years, five fentanyl analogs, carfentanil, furanyl fentanyl, acetyl fentanyl, 4-fluoroisobutyryl fentanyl (4-FIBF), and cyclopropyl fentanyl (CPrF), gained popularity and were found in 76.4% of the fentanyl analogs trafficked. In this study, we focused on 4-FIBF, CPrF, and structurally similar fentanyl analogs. We developed methods to differentiate these fentanyl analogs using theoretical and experimental methods. To do this, a set of fentanyl analogs were examined using density functional theory (DFT) calculations. The DFT results obtained in this project permitted the assignment of spectral bands. These results were then compared with normal Raman and SERS techniques. Structurally similar fentanyl analogs show important differences in their spectra, and they have been visually differentiated from each other both theoretically and experimentally. Additional results using principal component analysis and soft independent modeling of class analogy show they can be distinguished using this technique. The limit of detection values for FIBF and CPrF were determined to be 0.35 ng/mL and 4.4 ng/mL, respectively, using SERS. Experimental results obtained in this project can be readily implemented in field applications and smaller laboratories, where inexpensive portable Raman spectrometers are often present and used in drug analysis.

Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

An examination of differences in epigenetic methylation of saliva type samples based on collection method.

In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in-house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

A study of the potential application of digital PCR in the detection of fecal contamination of strawberries using Bacteroides markers.

Food-borne illnesses can result from contamination of agricultural products. In this study, we examined nanoplate digital PCR (dPCR) to test for fecal contamination of agricultural products. In nanoplate technique, the PCR mastermix is divided into 8.526,000 partitions, providing direct detection of individual DNA molecules, with correction by Poisson distribution. In this project, strawberries were inoculated with fecal material from animals, and the result detected by nanoplate digital PCR. A detection limit of 250 fg/uL was determined. Overall, dPCR offers a quick and sensitive method to detect contaminated produce.

Pyrosequencing: Current forensic methodology and future applications-a review.

The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.

High-resolution melt analysis for the detection of skin/sweat via DNA methylation.

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.

Development of a microwave-based extraction for forensic biological samples.

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

A collaborative exercise on DNA methylation-based age prediction and body fluid typing.

DNA methylation has become one of the most useful biomarkers for age prediction and body fluid identification in the forensic field. Therefore, several assays have been developed to detect age-associated and body fluid-specific DNA methylation changes. Among the many methods developed, SNaPshot-based assays should be particularly useful in forensic laboratories, as they permit multiplex analysis and use the same capillary electrophoresis instrumentation as STR analysis. However, technical validation of any developed assays is crucial for their proper integration into routine forensic workflow. In the present collaborative exercise, two SNaPshot multiplex assays for age prediction and a SNaPshot multiplex for body fluid identification were tested in twelve laboratories. The experimental set-up of the exercise was designed to reflect the entire workflow of SNaPshot-based methylation analysis and involved four increasingly complex tasks designed to detect potential factors influencing methylation measurements. The results of body fluid identification from each laboratory provided sufficient information to determine appropriate age prediction methods in subsequent analysis. In age prediction, systematic measurement differences resulting from the type of genetic analyzer used were identified as the biggest cause of DNA methylation variation between laboratories. Also, the use of a buffer that ensures a high ratio of specific to non-specific primer binding resulted in changes in DNA methylation measurement, especially when using degenerate primers in the PCR reaction. In addition, high input volumes of bisulfite-converted DNA often caused PCR failure, presumably due to carry-over of PCR inhibitors from the bisulfite conversion reaction. The proficiency of the analysts and experimental conditions for efficient SNaPshot reactions were also important for consistent DNA methylation measurement. Several bisulfite conversion kits were used for this study, but differences resulting from the use of any specific kit were not clearly discerned. Even when different experimental settings were used in each laboratory, a positive outcome of the study was a mean absolute age prediction error amongst participant's data of only 2.7 years for semen, 5.0 years for blood and 3.8 years for saliva.

Developmental validation of SpeID: A pyrosequencing-based assay for species identification.

In crime scenes, biological exhibits are often human in origin, yet biological stains from other fauna may also be present at a crime scene, creating confusion during an investigation. Furthermore, identifying the source of a biological sample can be critical during an investigation. To identify the presence of biological material from non-human sources, it is common to use genetic markers within mitochondrial DNA such as cytochrome b, 16S rRNA, and 12S rRNA genes. This process usually requires DNA sequencing, a process that is neither quick nor easy. In general, a faster, more standardized method for species identification from tissue and body fluids is desirable.For this reason, we have developed a vertebrate specific real-time quantitation method that is followed by an automated pyrosequencing-based procedure that sequences a short fragment within the 12S rRNA gene. Using no more than 35 bases, the assay can distinguish between 32 different species commonly found in and around a household with a turnaround time of 6 h from extraction to sequencing. -Using this procedure, up to 48 samples can be run at a time without the need for expensive reagents or bioinformatic skills.

Multivariate Analysis Aided Surface-Enhanced Raman Spectroscopy (MVA-SERS) Multiplex Quantitative Detection of Trace Fentanyl in Illicit Drug Mixtures Using a Handheld Raman Spectrometer.

Recently there has been upsurge in reports that illicit seizures of cocaine and heroin have been adulterated with fentanyl. Surface-enhanced Raman spectroscopy (SERS) provides a useful alternative to current screening procedures that permits detection of trace levels of fentanyl in mixtures. Samples are solubilized and allowed to interact with aggregated colloidal nanostars to produce a rapid and sensitive assay. In this study, we present the quantitative determination of fentanyl in heroin and cocaine using SERS, using a point-and-shoot handheld Raman system. Our protocol is optimized to detect pure fentanyl down to 0.20 ± 0.06 ng/mL and can also distinguish pure cocaine and heroin at ng/mL levels. Multiplex analysis of mixtures is enabled by combining SERS detection with principal component analysis and super partial least squares regression discriminate analysis (SPLS-DA), which allow for the determination of fentanyl as low as 0.05% in simulated seized heroin and 0.10% in simulated seized cocaine samples.

Fentanyl-related substance scheduling as an effective drug control strategy.

Fentanyl is now the primary driver of the current opioid crisis. Fentanyl and its analogues are subject to the Controlled Substances Act of 1970, the Controlled Substances Analogue Enforcement Act of 1986 (Federal Analogue Act), state laws, international treaties, and the laws of foreign countries. The appearance of novel psychoactive substances led to further legislative developments in scheduling. New fentanyl analogues proliferated in a manner previously unseen since about 2016. Overdose deaths of these fentanyl analogues prompted the Drug Enforcement Administration to reactively emergency schedule each new fentanyl analogue as it appeared. The international community also acted. Finally, on February 6, 2018, a proactive temporary (emergency) class-wide scheduling of fentanyl-related substances was implemented based upon the fentanyl core structure to save lives. This action spurred a similar action in China. Fentanyl analogues fell dramatically in the marketplace, despite further increases in fentanyl itself. Congress temporarily extended this scheduling, but it will soon expire. Opposition to permanent class-wide was lodged due to concerns over law enforcement overreach, inadequate Health and Human Services input, and hindrance of research. This paper reaffirms the importance of a class-based scheduling strategy while also arguing for increased research of schedule I controlled substances.

Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology.

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22-70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated-NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5-2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

The development of miniSTRs as a method for high-speed direct PCR.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.

A data-driven, high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids.

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

Match statistics for sequence-based alleles in profiles from forensic PCR-mps kits.

The first autosomal sequence-based allele (aka SNP-STR haplotype) frequency database for forensic massively parallel sequencing (MPS) has been published, thereby removing one of the remaining barriers to implementing MPS in casework. The database was developed using a specific set of flank trim sites. If different trim sites or different kits with different primers are used for casework, then SNP-STR haplotypes may be detected that do not have frequencies in the database. We describe a procedure to address calculation of match probabilities when casework samples are generated using an MPS kit with different trim sites than those present in the relevant population frequency database. The procedure provides a framework for comparison of any MPS kit or database combination while also accommodating comparison of MPS and CE profiles.

The genital microbiome and its potential for detecting sexual assault.

Since its inception, the Human Microbiome Project (HMP) has provided key discoveries that can be applied to forensics, in addition to those of obvious medical value. Whether for postmortem interval estimation, geolocation, or human identification, there are many applications of the microbiome as an investigative lead for forensic casework. The human skin microbiome has shown great potential for use in studies of transfer and human identification, however there has been little focus on the genital microbiome, in particular penile skin which differs from other body sites. Our preliminary data on both the penile and vaginal microbiome demonstrates potential value in cases of sexual assault. In this study we describe genital microbial signatures based on the analysis of five male and five female genital samples and compare these results to those from longitudinal studies. Selected taxa, e.g., Gardnerella, Lactobacilli, Finegoldia, Peptoniphilus, and Anaerococci, are shown to be candidate constituents of the genital microbiome that merit investigation for use in sexual assault casework.

Development of DNA methylation markers for sperm, saliva and blood identification using pyrosequencing and qPCR/HRM.

Discrimination of body fluids can provide important information in the investigation of crime scenes. The goal of this project was to identify new sets of tissue specific differentially methylated regions (tDMRs) and develop assays that can be utilized for forensic discrimination of body fluids, in particular sperm, saliva and blood. In this study, a sample set containing semen with sperm, semen without sperm, buccal swabs, saliva (oral fluids), venous blood, menstrual blood, vaginal secretions, and sweat/skin samples were used to develop four assays. Two methods for the analysis of DNA methylation biomarkers were developed in this paper: pyrosequencing and quantitative PCR/high resolution melt (HRM) analysis. Using an epigenome wide association study, two markers, NMUR2 and UBE2U, were found to be specific for sperm, based on the fact that mean DNA methylation levels for semen (containing sperm cells) were significantly lower than mean DNA methylation levels of other body fluids. In addition, one marker (SA-6) was hypermethylated in saliva when compared to other body fluids. The assays developed for NMUR2, UBE2U and SA-6 markers can be applied in forensic tissue identification using both pyrosequencing and HRM analysis. Additionally, a set of CpG sites in the AHRR locus were hypomethylated in blood when compared to other tissues using pyrosequencing. However, this locus was not amenable to HRM analysis. Overall, this work demonstrates the discovery and application of tDMRs for forensic applications.

A four-channel paper microfluidic device with gold nanoparticles and aptamers for seized drugs.

In this note we describe a four-channel paper microfluidic device (µPAD) that utilizes gold nanoparticles (AuNPs) as colorimetric sensors and specifically tailored aptamers to capture a set of targeted drugs, including cocaine, codeine and methamphetamine. The design utilizes salt-induced aggregation of AuNPs to produce a color change that indicates the presence of target analytes. In the absence of the targets, the aptamers bind to AuNPs, preventing aggregation. This µPAD was developed by optimizing the concentrations of aggregating agents, AuNPs and aptamers. The resultant device is sensitive and specific, producing a positive result upon detection of the target analyte.