A method of identifying the blood contributor in mixture stains through detecting blood-specific mRNA polymorphism

In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture.     

A new set of DIP‐SNP markers for detection of unbalanced and degraded DNA mixtures

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP‐SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP‐SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP‐SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP‐SNPs in a Chinese Han population. The DIP‐SNPs were capable of detecting the minor contributor's allele in home‐made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 –10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP‐SNPs may serve as a valuable tool in detection of UDM in forensic medicine.     

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA‐based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross‐tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case‐specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

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.     

Forensic applicability of multi‐allelic InDels with mononucleotide homopolymer structures

Insertion/deletion polymorphisms (InDels), which possess the characteristics of low mutation rates and a short amplicon size, have been regarded as promising markers for forensic DNA analysis. InDels can be classified as bi‐allelic or multi‐allelic, depending on the number of alleles. Many studies have explored the use of bi‐allelic InDels in forensic applications, such as individual identification and ancestry inference. However, multi‐allelic InDels have received relatively little attention. In this study, InDels with 2–6 alleles and a minor allele frequency ≥0.01, in Chinese Southern Han (CHS), were retrieved from the 1000 Genomes Project Phase III. Based on the structural analysis of all retrieved InDels, 17 multi‐allelic markers with mononucleotide homopolymer structures were selected and combined in one multiplex PCR reaction system. Sensitivity, species specificity and applicability in forensic case work of the multiplex were analyzed. A total of 218 unrelated individuals from a Chinese Han population were genotyped. The combined discriminatory power (CDP), the combined match probability (CMP) and the cumulative probability of exclusion (CPE) were 0.9999999999609, 3.91E‐13 and 0.9956, respectively. The results demonstrated that this InDel multiplex panel was highly informative in the investigated population and most of the 26 populations of the 1000 Genomes Project. The data also suggested that multi‐allelic InDel markers with monomeric base pair expansions are useful for forensic applications.     

Electrochemical Biosensing in Cancer Diagnostics and Follow‐up

In cancer, screening and early detection are critical for the success of the patient's treatment and to increase the survival rate. The development of analytical tools for non‐invasive detection, through the analysis of cancer biomarkers, is imperative for disease diagnosis, treatment and follow‐up. Tumour biomarkers refer to substances or processes that, in clinical settings, are indicative of the presence of cancer in the body. These biomarkers can be detected using biosensors, that, because of their fast, accurate and point of care applicability, are prominent alternatives to the traditional methods. Moreover, the constant innovations in the biosensing field improve the determination of normal and/or elevated levels of tumour biomarkers in patients’ biological fluids (such as serum, plasma, whole blood, urine, etc.). Although several biomarkers (DNA, RNA, proteins, cells) are known, the detection of proteins and circulating tumour cells (CTCs) are the most commonly reported due to their approval as tumour biomarkers by the specialized entities and commonly accepted for diagnosis by medical and clinical teams. Therefore, electrochemical immunosensors and cytosensors are vastly described in this review, because of their fast, simple and accurate detection, the low sample volumes required, and the excellent limits of detection obtained. The biosensing strategies reported for the six most commonly diagnosed cancers (lung, breast, colorectal, prostate, liver and stomach) are summarized and the distinct phases of the sensors’ constructions (surface modification, antibody immobilization, immunochemical interactions, detection approach) and applications are discussed.     

Wear behavior of light‐cured dental composite reinforced with silane‐treated nanosilica filler

In this study, the effect of varying different weight fraction of silane‐treated nanosilica (0‐15 wt%) on the wear behavior of Bisphenol‐A glycidyl methacrylate/tri‐ethylene glycol dimethacrylate–based dental composite was analyzed. Fourier transform infrared spectroscopy, transmission electron microscope, and thermo‐gravimetric analysis were used to characterize silane‐treated filler. The in vitro wear tests were performed up to 20 000 cycles using dental wear simulator. Four different working conditions were discussed including 2‐body wear in distilled water and artificial saliva as well as 3‐body wear in slurry of poppy seed mixed in distilled water and poppy seed mixed in artificial saliva. The results suggested that composites with increased in nanosilica fillers exhibited lower wear volume and smoother worn surface in all working mediums. In 2‐body abrasive wear, the wear rate in distilled water was 10.05% more than that in artificial saliva condition. However, in 3‐body abrasive wear, the wear rate in slurry of poppy seed mixed in artificial saliva was 15.96% more than that in the medium of poppy seed mixed in distilled water condition. Also, the 2‐body abrasive wear rate was 56% and 22% more than the 3‐body abrasive wear rate in the slurry of distilled water and artificial saliva condition, respectively.     

A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip‐based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads‐based “sandwich” hybridization strategy, was developed for the sensitive and ultra‐specific detection of single‐base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single‐base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer‐related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10^?11 M, a RSD (n = 5) < 5% and a discrimination factor of 3.58–4.54 for one‐base mismatch.     

Two‐step signal amplification for high‐sensitivity detection of biomarkers using gold nanoparticle–based conjugates

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)‐based, two‐step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96‐well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)‐conjugated detection antibodies were labeled on large‐size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high‐sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate‐specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.     

A novel method for the analysis of 20 multi‐Indel polymorphisms and its forensic application

Insertion/deletion polymorphisms (Indels) have been considered as potential markers for forensic DNA analysis. However, the discrimination power of Indels is relatively lower due to the poor polymorphisms of diallelic markers. Here, two to three Indel loci that were very tightly linked in physical position were combined into a specific multi‐Indel marker to improve the discrimination, as well as a multiplex that consisted of a set of multi‐Indel markers was developed for forensic purpose. Finally, a multiplex system with 20 multi‐Indel markers including 43 Indel loci from dbSNP database was constructed and DNA sample can be analyzed by this multiplex in one PCR reaction and one CE run. A total of 150 unrelated individuals from Hunan province in South‐central China were genotyped by the multiplex system. The result showed that a total of 63 specific amplicons were detected, three alleles were observed in multi‐Indel markers including two Indel loci and four alleles were observed in the markers including three Indel loci. The cumulative probability of exclusion and the accumulated discrimination power were 0.9989 and 0.9999999999994, respectively. Our result demonstrated that the strategy could be efficient to develop higher polymorphic multi‐Indel markers, and the new multiplex could provide Supporting Information for forensic application.     

A G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence assay for highly sensitive detection of biotin in flour

Quantitative analysis of biotin in biological fluids, foods, and pharmaceutical is important for diagnosis and treatment of biotin‐related diseases and health maintenance. In this work, a novel G‐quadruplex/hemin DNAzyme‐based microchip electrophoresis chemiluminescence (CL) assay method was established for rapid and highly sensitive detection of biotin. This method is based on the specific binding between biotin and streptavidin, the catalytic CL characteristics of G‐quadruplex/hemin DNAzyme to the oxidation–reduction reaction of hydrogen peroxide with luminol, and the on‐line separation function of microchip electrophoresis. Under the optimal experimental conditions, on‐chip biotin analysis was achieved within 1 min. The CL intensity is linearly proportional to the concentration of biotin in the range of 13–630 nM with a detection limit of 6.4 nM. The proposed method has been applied for the detection of biotin in flour, biotin contents in three flour samples are found in the range of 199–223 ng/g with a mean value of 214 ng/g. The recoveries were in the range of 94–103%. With excellent sensitivity and good selectivity, the proposed method could be applied in a wide range of biological fluids, foods, and pharmaceutical analysis.     

Toward multimarker and functional assays from crude cell lysates: controlling spacing and signal amplification in DNA-CT–based bioelectrochemical devices

DNA-based electrochemical biosensors that rely on charge transport through DNA (DNA-CT) to detect biomarkers of interest have shown great promise in proof-of-principle studies because of their specificity, sensitivity, and low cost. However, this approach has not translated successfully into real clinical applications. One significant barrier has been the need to measure biomarkers in unpurified, complex cell and tissue lysates, which is difficult with DNA-modified electrodes because of crowding and nonspecific adhesion of biomolecules to the surface. Here, we discuss recent achievements in the control of DNA monolayer formation and the amplification of DNA CT signals that help to facilitate the detection of meaningful biomarkers from unprocessed, clinically relevant lysate samples.     

Simultaneous Quantification of Multiple Cancer Biomarkers in Blood Samples through DNA‐Assisted Nanopore Sensing

Protein biomarkers in blood have been widely used in the early diagnosis of disease. However, simultaneous detection of many biomarkers in a single sample remains challenging. Herein, we show that the combination of a sandwich assay and DNA‐assisted nanopore sensing could unambiguously identify and quantify several antigens in a mixture. We use five barcode DNAs to label different gold nanoparticles that can selectively bind specific antigens. After the completion of the sandwich assay, barcode DNAs are released and subject to nanopore translocation tests. The distinct current signatures generated by each barcode DNA allow simultaneous quantification of biomarkers at picomolar level in clinical samples. This approach would be very useful for accurate and multiplexed quantification of cancer‐associated biomarkers within a very small sample volume, which is critical for non‐invasive early diagnosis of cancer.     

Multiplex identification of drug‐resistant Gram‐positive pathogens using stuffer‐free MLPA system

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood.     

Development of a solid‐phase microextraction LC–MS/MS method for determination of oxidative stress biomarkers in biofluids

Recently the connection between oxidative stress and various diseases, including cancer and Alzheimer's, attracts notice as a pathway suitable for diagnostic purposes. 8‐Oxo‐deoxyguanosine and 8‐oxo‐deoxyadenosine produced from the interaction of reactive oxygen species with DNA become prominent as biomarkers. Several methods have been developed for their determination in biofluids, including solid‐phase extraction and enzyme‐linked immunosorbent assays. However, still, there is a need for reliable and fast analytical methods. In this context, solid‐phase microextraction offers many advantages such as flexibility in geometry and applicable sample volume, as well as high adaptability to high‐throughput sampling. In this study, a solid‐phase microextraction method was developed for the determination of 8‐oxo‐deoxyguanosine and 8‐oxo‐deoxyadenosine in biofluids. The extractive phase of solid‐phase microextraction consisted of hydrophilic–lipophilic balanced polymeric particles. In order to develop a solid‐phase microextraction method suitable for the determination of the analytes in saliva and urine, several parameters, including desorption solvent, desorption time, sample pH, and ionic strength, were scrutinized. Analytical figures of merit indicated that the developed method provides reasonable interday and intraday precisions (<15% in both biofluids) with acceptable accuracy. The method provides a limit of quantification for both biomarkers at 5.0 and 10.0 ng/mL levels in saliva and urine matrices, respectively.     

Development of a body fluid identification multiplex via DNA methylation analysis

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers – BCAS4, CG06379435, VE_8, and ZC3H12D – were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.     

Developing a multiplex mtSNP assay for forensic application in Han Chinese based on mtDNA phylogeny and hot spot

We designed one multiplex assay with a reduced number of SNPs from whole mitochondrial genome as a screening approach for forensic purposes and developed a multiplex SNaPshot assay with 26 mitochondrial SNPs (mtSNPs). This assay included 16 target mtSNPs that defined the main haplogroups in Chinese population and ten hot‐spot mtSNPs found by pyrosequencing. To validate our multiplex mtSNP assay, we not only analyzed a Chinese Han population sample, but also sequenced the complete control region of same set of individuals. Fifty‐one haplotypes were observed in 204 individuals using our multiplex mtSNP assay and the haplotype diversity was estimated to be 0.9626. Our multiplex mtSNP assay could also distinguish some individuals sharing the same control region sequences. The same mtSNP profiles were obtained from the bloodstain, hair shaft, and salivary swab from same individuals. A good profile could be obtained with 50 pg of DNA. It was evident that our multiplex mtSNP assay not only improved the discrimination power, but also allowed allocating mitochondrial DNA profiles to particular haplogroups not clearly defined with the control region alone. We highlight the importance of the balance of target mtSNPs for haplogroup assignment and hot‐spot mtSNPs for increasing discrimination power.     

A Rapid,Amplification‐Free,and Sensitive Diagnostic Assay for Single‐Step Multiplexed Fluorescence Detection of MicroRNA

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short‐sequence nucleic acids next‐generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single‐step, single‐temperature, and amplification‐free assay provides a unique combination of performance parameters compared to state‐of‐the‐art miRNA detection technologies. Precise multiplexed quantification of miRNA‐20a, ‐20b, and ‐21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high‐throughput and point‐of‐care clinical diagnostics.