KLK7 expression in human tumors: a tissue microarray study on 13,447 tumors.

BACKGROUND: Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells. METHODS: To comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: KLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p = 0.0005), blood vessel infiltration (p = 0.0037), and lymph vessel infiltration (p < 0.0001) in colorectal adenocarcinoma, nodal metastasis in hepatocellular carcinoma (p = 0.0382), advanced pathological tumor stage in papillary thyroid cancer (p = 0.0132), and low grade of malignancy in a cohort of 719 squamous cell carcinomas from 11 different sites of origin (p < 0.0001). CONCLUSIONS: These data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated.

Matrix-assisted laser desorption/ionization mass spectrometry imaging as a new tool for molecular histopathology in epilepsy surgery.

OBJECTIVE: Epilepsy surgery is a treatment option for patients with seizures that do not respond to pharmacotherapy. The histopathological characterization of the resected tissue has an important prognostic value to define postoperative seizure outcome in these patients. However, the diagnostic classification process based on microscopic assessment remains challenging, particularly in the case of focal cortical dysplasia (FCD). Imaging mass spectrometry is a spatial omics technique that could improve tissue phenotyping and patient stratification by investigating hundreds of biomolecules within a single tissue sample, without the need for target-specific reagents. METHODS: An in situ proteomic technique called matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is here investigated as a potential new tool to expand conventional diagnosis on standard paraffin brain tissue sections. Unsupervised and region of interest-based MALDI-MSI analyses of sections from 10 FCD type IIb (FCDIIb) cases were performed, and the results were validated by immunohistochemistry. RESULTS: MALDI-MSI identified distinct histopathological features and the boundaries of the dysplastic lesion. The capability to visualize the spatial distribution of well-known diagnostic markers enabling multiplex measurements on single tissue sections was demonstrated. Finally, a fingerprint list of potential discriminant peptides that distinguish FCD core from peri-FCD tissue was generated. SIGNIFICANCE: This is the first study that explores the potential application of MALDI-MSI in epilepsy postsurgery fixed tissue, by utilizing the well-characterized FCDIIb features as a model. Extending these preliminary analyses to a larger cohort of patients will generate spectral libraries of molecular signatures that discriminate tissue features and will contribute to patient phenotyping.

Sr-Pb isotope differences in pre- and post-burial human bone, teeth, and hair keratin: implications for isotope forensics.

The isotopic signatures of human tissues can provide valuable information on geographic origin for medicolegal investigations involving unidentified persons. It is important to understand the impact of diagenetic processes on isotopic signatures, as alterations could result in incorrect estimation of geographic origin. This study examines alterations in isotope signatures of different tissues of five human body donors studied throughout decomposition at the Forensic Anthropology Research Facility (FARF), San Marcos, TX. Two body donors were buried, two were placed in open pits, and one was first allowed to naturally mummify and then buried. Remains were recovered after a period of 7-34 months. The preplacement and post-recovery Sr-Pb isotope data of scalp hair, bone (iliac and tibia), and tooth enamel and dentine were compared. The hair samples record significant shifts in Sr-Pb isotope compositions, with hair keratin Pb isotope composition shifting towards the Pb signature of local soil samples. Hair keratin Sr isotope compositions were altered by the burial environment and possibly also by the lab sample cleaning method. The spongy iliac bone samples show inconsistencies in the recoverability of the preplacement Sr-Pb isotope signatures. The post-placement signatures of the buried donors show slight elevation over preplacement signatures. The post-placement signatures of donors placed in open pits are significantly elevated. The tibia and dental samples record the most consistent isotopic data with the least alteration. These more densely mineralised elements show good recoverability of the preplacement isotope signatures in burials and open pits and are thus deemed better targets for forensic investigative purposes.

Evaluation of distribution of emerging mycotoxins in human tissues: applications of dispersive liquid-liquid microextraction and liquid chromatography-mass spectrometry.

In this work, a complete study of the distribution of emerging mycotoxins in the human body has been carried out. Specifically, the presence of enniatins (A, A1, B, B1) and beauvericin has been monitored in brain, lung, kidney, fat, liver, and heart samples. A unique methodology based on solid-liquid extraction (SLE) followed by dispersive liquid-liquid microextraction (DLLME) was proposed for the six different matrices. Mycotoxin isolation was performed by adding ultrapure water, acetonitrile, and sodium chloride to the tissue sample for SLE, while the DLLME step was performed using chloroform as extraction solvent. Subsequently, the analysis was carried out by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The proposed method allowed limits of quantification (LOQs) to be obtained in a range of 0.001-0.150 ng g-1, depending on the tissue and mycotoxin. The precision was investigated intraday and interday, not exceeding of 9.8% of relative standard deviation. In addition, trueness studies achieved 75 to 115% at a mycotoxin concentration of 25 ng g-1 and from 82 to 118% at 5 ng g-1. The application of this methodology to 26 forensic autopsies demonstrated the bioaccumulation of emerging mycotoxins in the human body since all mycotoxins were detected in tissues. Enniatin B (ENNB) showed a high occurrence, being detected in 100% of liver (7 ± 13 ng g-1) and fat samples (0.2 ± 0.8 ng g-1). The lung had a high incidence of all emerging mycotoxins at low concentrations, while ENNB, ENNB1, and ENNA1 were not quantifiable in heart samples. Co-occurrence of mycotoxins was also investigated, and statistical tests were applied to evaluate the distribution of these mycotoxins in the human body.

Discerning animal-sourced food in diet using isotope analysis of human scalp hair and fingernails.

PURPOSE: Diet-related diseases are advancing as the leading cause of death globally. As self-reporting of diet by patients can be associated with errors, stable isotopes of human tissues can be used to diagnose diseases, understand physiology, and detect change in diet. This study investigates the effect of type and amount of food on the nitrogen and carbon concentration (Nconc and Cconc) and isotopic composition (δ15N and δ13C) in human scalp hair and fingernails. METHODS: A total of 100 residents participated in the study whereas only 74 individuals provided complete diet history. Sixty-six food items majorly available to them were also collected. The Nconc, Cconc, δ15N and δ13C values of human hair, nails and food items were determined. RESULTS: The Nconc, Cconc, δ15N and δ13C values between plant-sourced and animal-sourced food items, as well as human hair and nail tissue were significantly different (p < 0.05). The δ15N value of human tissues was distinct between lacto-vegetarians and omnivores by 0.9‰. The δ15N and δ13C values of human tissues increased by 0.4-0.5‰ with every 5% increase in the consumption of animal protein. CONCLUSIONS: The study helps to demarcate lacto-vegetarians from omnivores, and estimate the percentage of animal protein in diet based on the dual isotope values of human tissues. It also acts as a reference to determine isotopic composition of hair tissue provided the isotope value of nail tissue is known and vice versa.

Potential of in vitro nuclear magnetic resonance of biofluids and tissues in clinical research.

Body fluids, cells, and tissues contain a wide variety of metabolites that consist of a mixture of various low-molecular-weight compounds, including amino acids, peptides, lipids, nucleic acids, and organic acids, which makes comprehensive analysis more difficult. Quantitative nuclear magnetic resonance (NMR) spectroscopy is a well-established analytical technique for analyzing the metabolic profiles of body fluids, cells, and tissues. It enables fast and comprehensive detection, characterization, a high level of experimental reproducibility, minimal sample preparation, and quantification of various endogenous metabolites. In recent times, NMR-based metabolomics has been appreciably utilized in diverse branches of medicine, including microbiology, toxicology, pathophysiology, pharmacology, nutritional intervention, and disease diagnosis/prognosis. In this review, the utility of NMR-based metabolomics in clinical studies is discussed. The significance of in vitro NMR-based metabolomics as an effective tool for detecting metabolites and their variations in different diseases are discussed, together with the possibility of identifying specific biomarkers that can contribute to early detection and diagnosis of disease.

CC chemokine ligand 20 (CCL20) positively regulates collagen type I production in 3D skin equivalent tissues.

Chemokines are a group of small proteins that induce chemoattraction and inflammation and contribute to the differentiation and homeostasis of various cell types. Here we explored the role of chemokines, extracellular matrix production, and myofibroblast differentiation in self-assembled skin equivalents (SASE), a three-dimensional (3D) skin-equivalent tissue model. We found that the expression of three chemokines, C-C motif chemokine ligand (CCL) 20, C-X-C motif chemokine ligand (CXCL) 5, and CXCL8, increased with differentiation to myofibroblasts. Addition of recombinant CCL20 to human skin fibroblast induced collagen Type I alpha 2 gene expression, but did not affect the expression of alpha smooth muscle actin expression. Conversely, siRNA gene knockdown of CCL20 effectively inhibited the expression of collagen Type I gene and protein. Furthermore, when the CCL20 gene in fibroblasts was knocked down in SASE, collagen Type I synthesis and stromal thickness were decreased. Taken together, these results have indicated the utility of SASE in understanding how cytokines such as CCL20 positively regulate extracellular matrix proteins such as collagen Type I production during myofibroblast differentiation in 3D tissues that mimic human skin.

Human biomonitoring of microplastics and health implications: A review.

BACKGROUND: Microplastics (MPs) are plastic particles (<5 mm) ubiquitous in water, soil, and air, indicating that humans can be exposed to MPs through ingestion of water and food, and inhalation. OBJECTIVE: This review provides an overview of the current human biomonitoring data available to evaluate human exposure and health impact of MPs. METHOD: We compiled 91 relevant studies on MPs in human matrices and MPs toxicological endpoints to provide evidence on MPs distribution in the different tissues and the implications this can have from a health perspective. RESULTS: Human exposure to MPs has been corroborated by the detection of MPs in different human biological samples including blood, urine, stool, lung tissue, breast milk, semen and placenta. Although humans have clearance mechanisms protecting them from potentially harmful substances, health risks associated to MPs exposure include the onset of inflammation, oxidative stress, and DNA damage, potentially leading to cardiovascular and respiratory diseases, as well as cancer, as suggested by in vitro and in vivo studies. CONCLUSION: Based on compiled data, MPs have been recurrently identified in different human tissues and fluids, suggesting that humans are exposed to MPs through inhalation and ingestion. Despite differences in MPs concentrations appear in exposed and non-exposed people, accumulation and distribution pathways and potential human health hazards is still at an infant stage. Human biomonitoring data enables the assessment of human exposure to MPs and associated risks, and this information can contribute to draw management actions and guidelines to minimize MP release to the environment, and thus, reduce human uptake.

Monitoring of hydroxylated polycyclic aromatic hydrocarbons in human tissues: Targeted and untargeted approaches using liquid chromatography-high resolution mass spectrometry.

Hydroxylated polycyclic aromatic hydrocarbons are metabolites of persistent organic pollutants which are formed during the bioactivation process of biological matrices and whose toxicity is being studied. The aim of this work was the development of a novel analytical method for the determination of these metabolites in human tissues, known to have bioaccumulated their parent compounds. Samples were treated by salting-out assisted liquid-liquid extraction and the extracts were analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry with a hybrid quadrupole-time-of-flight analyzer. The proposed method achieved limits of detection in the 0.15-9.0 ng/g range for the five target analytes (1-hydroxynaphthalene, 1-hydroxypyrene, 2-hydroxynaphthalene, 7-hydroxybenzo[a]pyrene, and 9-hydroxyphenanthrene). The quantification was achieved by matrix-matched calibration using 2,2´-biphenol as internal standard. For all compounds, relative standard deviation, calculated for six successive analyses, was below 12.1%, demonstrating good precision for the developed method. None of the target compounds was detected in the 34 studied samples. Moreover, an untargeted approach was applied to study the presence of other metabolites in the samples, as well as their conjugated forms and related compounds. For this objective, a homemade mass spectrometry database covering 81 compounds was created and none of them was detected in the samples.

The indigenous African cultural value of human tissues and implications for bio-banking.

Bio-banking in research elicits numerous ethical issues related to informed consent, privacy and identifiability of samples, return of results, incidental findings, international data exchange, ownership of samples, and benefit sharing etc. In low and middle income (LMICs) countries the challenge of inadequate guidelines and regulations on the proper conduct of research compounds the ethical issues. In addition, failure to pay attention to underlying indigenous worldviews that ought to inform issues, practices and policies in Africa may exacerbate the situation. In this paper we discuss how the African context presents unique and outstanding cultural thought systems regarding the human body and biological materials that can be put into perspective in bio-bank research. We give the example of African ontology of nature presented by John Samwel Mbiti as foundational in adding value to the discourse about enhancing relevance of bio-bank research in the African context. We underline that cultural rites of passage performed on the human body in majority of communities in Africa elicit quintessential perspective on beliefs about handling of human body and human biological tissues. We conclude that acknowledgement and inclusion of African indigenous worldviews regarding the human body is essential in influencing best practices in biobank research in Africa.

Stage-specific differential expression of zinc transporter SLC30A and SLC39A family proteins during prostate tumorigenesis.

Prostate cancer (PCa) initiation and progression uniquely modify the prostate milieu to aid unrestrained cell proliferation. One salient modification is the loss of the ability of prostate epithelial cells to accumulate high concentrations of zinc; however, molecular alterations associated with loss of zinc accumulating capability in malignant prostate cells remain poorly understood. Herein, we assessed the stage-specific expression of zinc transporters (ZNTs) belonging to the ZNT (SLC30A) and Zrt- and Irt-like protein (ZIP) (SLC39A) solute-carrier family in the prostate tissues of different genetically engineered mouse models (GEMM) of PCa (TMPRSS2-ERG.Ptenflox/flox , Hi-Myc+/- , and transgenic adenocarcinoma of mouse prostate), their age-matched wild-type controls, and 104 prostate core biopsies from human patients with different pathological lesions. Employing immunohistochemistry, differences in the levels of protein expression and spatial distribution of ZNT were evaluated as a function of the tumor stage. Results indicated that the expression of zinc importers (ZIP1, ZIP2, and ZIP3), which function to sequester zinc from circulation and prostatic fluid, was low to negligible in the membranes of the malignant prostate cells in both GEMM and human prostate tissues. Regarding zinc exporters (ZNT1, ZNT2, ZNT9, and ZNT10) that export excess zinc into the extracellular spaces or intracellular organelles, their expression was low in normal prostate glands of mice and humans; however, it was significantly upregulated in prostate adenocarcinoma lesions in GEMM and PCa patients. Together, our findings provide new insights into altered expression of ZNTs during the progression of PCa and indicate that changes in zinc homeostasis could possibly be an early-initiation event during prostate tumorigenesis and a likely prevention/intervention target.

Better governance starts with better words: why responsible human tissue research demands a change of language.

The rise of precision medicine has led to an unprecedented focus on human biological material in biomedical research. In addition, rapid advances in stem cell technology, regenerative medicine and synthetic biology are leading to more complex human tissue structures and new applications with tremendous potential for medicine. While promising, these developments also raise several ethical and practical challenges which have been the subject of extensive academic debate. These debates have led to increasing calls for longitudinal governance arrangements between tissue providers and biobanks that go beyond the initial moment of obtaining consent, such as closer involvement of tissue providers in what happens to their tissue, and more active participatory approaches to the governance of biobanks. However, in spite of these calls, such measures are being adopted slowly in practice, and there remains a strong tendency to focus on the consent procedure as the tool for addressing the ethical challenges of contemporary biobanking. In this paper, we argue that one of the barriers to this transition is the dominant language pervading the field of human tissue research, in which the provision of tissue is phrased as a 'donation' or 'gift', and tissue providers are referred to as 'donors'. Because of the performative qualities of language, the effect of using 'donation' and 'donor' shapes a professional culture in which biobank participants are perceived as passive providers of tissue free from further considerations or entitlements. This hampers the kind of participatory approaches to governance that are deemed necessary to adequately address the ethical challenges currently faced in human tissue research. Rather than reinforcing this idea through language, we need to pave the way for the kind of participatory approaches to governance that are being extensively argued for by starting with the appropriate terminology.

The Use of Human Tissues for Research: What Investigators Need to Know.

While laboratory animals are necessary for some aspects of the development of scientific and biomedical advances, including those of precision medicine, the use of human tissues is necessary in order to explore the findings and ensure that they are relevant to human systems. Many sources of human tissues exist, but researchers - particularly those making the transition from animal to human systems - may not be aware of how best to find quality sources of human tissues or how best to use them in their research. In this article, we discuss the advantages of using human tissues in research. In addition, we highlight some of the major advances made possible through the use of human tissue, and describe how human tissue is collected for research. We discuss the various types of bioresources that make human tissue available, and advise on how investigators can find and use appropriate bioresources to support their research - with the hope that this information will help facilitate the transition from research on animals to research using human tissues, as rapidly as is practicable.

Iron-oxide minerals in the human tissues.

Iron is critically important and highly regulated trace metal in the human body. However, in its free ion form, it is known to be cytotoxic; therefore, it is bound to iron storing protein, ferritin. Ferritin is a key regulator of body iron homeostasis able to form various types of minerals depending on the tissue environment. Each mineral, e.g. magnetite, maghemite, goethite, akaganeite or hematite, present in the ferritin core carry different characteristics possibly affecting cells in the tissue. In specific cases, it can lead to disease development. Widely studied connection with neurodegenerative conditions is widely studied, including Alzheimer disease. Although the exact ferritin structure and its distribution throughout a human body are still not fully known, many studies have attempted to elucidate the mechanisms involved in its regulation and pathogenesis. In this review, we try to summarize the iron uptake into the body. Next, we discuss the known occurrence of ferritin in human tissues. Lastly, we also examine the formation of iron oxides and their involvement in brain functions.

A comprehensive investigation of the mRNA and protein level of ACE2, the putative receptor of SARS-CoV-2, in human tissues and blood cells.

The outbreak of pneumonia caused by SARS-CoV-2 posed a great threat to global human health, which urgently requires us to understand comprehensively the mechanism of SARS-CoV-2 infection. Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for SARS-CoV-2, distribution of which may indicate the risk of different human organs vulnerable to SARS-CoV-2 infection. Previous studies investigating the distribution of ACE2 mRNA in human tissues only involved a limited size of the samples and a lack of determination for ACE2 protein. Given the heterogeneity among humans, the datasets covering more tissues with a larger size of samples should be analyzed. Indeed, ACE2 is a membrane and secreted protein, while the expression of ACE2 in blood and common blood cells remains unknown. Herein, the proteomic data in HIPED and the antibody-based immunochemistry result in HPA were collected to analyze the distribution of ACE2 protein in human tissues. The bulk RNA-seq profiles from three separate public datasets including HPA tissue Atlas, GTEx, and FANTOM5 CAGE were also obtained to determine the expression of ACE2 in human tissues. Moreover, the abundance of ACE2 in human blood and blood cells was determined by analyzing the data in the PeptideAtlas and the HPA Blood Atlas. We found that the mRNA expression cannot reflect the abundance of ACE2 factor due to the strong differences between mRNA and protein quantities of ACE2 within and across tissues. Our results suggested that ACE2 protein is mainly expressed in the small intestine, kidney, gallbladder, and testis, while the abundance of which in brain-associated tissues and blood common cells is low. HIPED revealed enrichment of ACE2 protein in the placenta and ovary despite a low mRNA level. Further, human secretome shows that the average concentration of ACE2 protein in the plasma of males is higher than those in females. Our research will be beneficial for understanding the transmission routes and sex-based differences in susceptibility of SARS-CoV-2 infection.

Joint analyses of multi-tissue Hi-C and eQTL data demonstrate close spatial proximity between eQTLs and their target genes.

BACKGROUND: Gene regulation is important for cells and tissues to function. It has been studied from two aspects at the genomic level, the identification of expression quantitative trait loci (eQTLs) and identification of long-range chromatin interactions. It is important to understand their relationship, such as whether eQTLs regulate their target genes through physical chromatin interaction. Although chromatin interactions have been widely believed to be one of the main mechanisms underlying eQTLs, most evidence came from studies of cell lines and yet no direct evidence exists for tissues. RESULTS: We performed various joint analyses of eQTL and high-throughput chromatin conformation capture (Hi-C) data from 11 human primary tissue types and 2 human cell lines. We found that chromatin interaction frequency is positively associated with the number of genes that have eQTLs and that eQTLs and their target genes tend to fall into the same topologically associating domain (TAD). These results are consistent across all tissues and cell lines we evaluated. Moreover, in 6 out of 11 tissues (aorta, dorsolateral prefrontal cortex, hippocampus, pancreas, small bowel, and spleen), tissue-specific eQTLs are significantly enriched in tissue-specific frequently interacting regions (FIREs). CONCLUSIONS: Our data have demonstrated the close spatial proximity between eQTLs and their target genes among multiple human primary tissues.

Mapping human N-linked glycoproteins and glycosylation sites using mass spectrometry.

N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.

Correlation and Similarity between Cerebral and Non-Cerebral Electrical Activity for User's States Assessment.

Human tissues own conductive properties, and the electrical activity produced by human organs can propagate throughout the body due to neuro transmitters and electrolytes. Therefore, it might be reasonable to hypothesize correlations and similarities between electrical activities among different parts of the body. Since no works have been found in this direction, the proposed study aimed at overcoming this lack of evidence and seeking analogies between the brain activity and the electrical activity of non-cerebral locations, such as the neck and wrists, to determine if i) cerebral parameters can be estimated from non-cerebral sites, and if ii) non-cerebral sensors can replace cerebral sensors for the evaluation of the users under specific experimental conditions, such as eyes open or closed. In fact, the use of cerebral sensors requires high-qualified personnel, and reliable recording systems, which are still expensive. Therefore, the possibility to use cheaper and easy-to-use equipment to estimate cerebral parameters will allow making some brain-based applications less invasive and expensive, and easier to employ. The results demonstrated the occurrence of significant correlations and analogies between cerebral and non-cerebral electrical activity. Furthermore, the same discrimination and classification accuracy were found in using the cerebral or non-cerebral sites for the user's status assessment.

Collection and Preparation of Clinical Samples for Metabolomics.

A wide range of biofluids (urine, serum, plasma, saliva, etc.) as well as cellular and tissue samples can be collected and investigated in clinical metabolomic studies. The choice of sample is dependent on the clinical question being investigated with biofluids typically studied to identify biomarkers, whereas tissues and primary/immortalised cells are typically studied to investigate mechanisms associated with pathophysiological processes. Methods applied to collect samples, quench metabolism and extract samples differ between sample types from simple collect, dilute and analyse methods for urine to complex washing, quenching and biphasic extraction methods for tissues. The range of sample collection and extraction methods are discussed with sample-specific considerations highlighted. Finally, methods for imaging of cells and tissues and for in vivo metabolomic analysis will also be introduced.

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 µL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNAt) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.