RNA capture pin technology: investigating long-term stability and mRNA purification specificity of oligonucleotide immobilization on gold and streptavidin surfaces.

Advancing biomedical studies necessitates the development of cutting-edge technologies for the rapid extraction of nucleic acid. We characterized an RNA capture pin (RCP) tool that is non-destructive to the sample and enables rapid purification and enrichment of mRNA for subsequent genetic analysis. At the core of this technology is a pin (200 µm × 3 cm) functionalized with dT15 capture sequences that hybridize to mRNA within 2 min of insertion in the specimen. Two methods for immobilizing the oligos on the surface of the RCPs were investigated: gold-thiol and biotin-streptavidin. The RNA capture efficiency of the RCPs was assessed using a radish plant. The average reverse transcription-quantitative polymerase chain reaction (RT-qPCR) cycle amplification values were 19.93 and 24.84 for gold- and streptavidin-coated pins, respectively. The amount of RNA present on the surface of the probes was measured using the Agilent 2100 Bioanalyzer. RNA sequencing was performed to determine the mRNA selectivity of the RNA capture pin. Gene read count analysis confirmed that the RNA purified via the gold-plated RCPs contained 70% messenger RNA, 10% ribosomal RNA, and 20% non-coding RNA. The long-term stability of the bond between the dT15 oligos and the surface of the RCPs was assessed over 4 months. A significant decrease in the dT15 surface coverage of the streptavidin-coated RCPs was observed after 2 weeks of storage at 4 °C. The gold-thiol RNA capture pins exhibited a retention rate of 40% of the oligos after 4 months of storage.

Biosensor Development and Innovation in Healthcare and Medical Applications.

The pandemic necessitated a change to the historical diagnostics model [...].

Advances in Biosensors Technology for Detection and Characterization of Extracellular Vesicles.

Exosomes are extracellular vehicles (EVs) that encapsulate genomic and proteomic material from the cell of origin that can be used as biomarkers for non-invasive disease diagnostics in point of care settings. The efficient and accurate detection, quantification, and molecular profiling of exosomes are crucial for the accurate identification of disease biomarkers. Conventional isolation methods, while well-established, provide the co-purification of proteins and other types of EVs. Exosome purification, characterization, and OMICS analysis are performed separately, which increases the complexity, duration, and cost of the process. Due to these constraints, the point-of-care and personalized analysis of exosomes are limited in clinical settings. Lab-on-a-chip biosensing has enabled the integration of isolation and characterization processes in a single platform. The presented review discusses recent advancements in biosensing technology for the separation and detection of exosomes. Fluorescent, colorimetric, electrochemical, magnetic, and surface plasmon resonance technologies have been developed for the quantification of exosomes in biological fluids. Size-exclusion filtration, immunoaffinity, electroactive, and acoustic-fluid-based technologies were successfully applied for the on-chip isolation of exosomes. The advancement of biosensing technology for the detection of exosomes provides better sensitivity and a reduced signal-to-noise ratio. The key challenge for the integration of clinical settings remains the lack of capabilities for on-chip genomic and proteomic analysis.

Therapeutic potential of astrocyte-derived extracellular vesicles in mitigating cytotoxicity and transcriptome changes in human brain endothelial cells.

This study investigates the therapeutic effect of astrocyte-derived extracellular vesicles (EVs) in mitigating neurotoxicity-induced transcriptome changes, mitochondrial function, and base excision repair mechanisms in human brain endothelial cells (HBECs). Neurodegenerative disorders are marked by inflammatory processes impacting the blood-brain barrier (BBB) that involve its main components- HBECs and astrocytes. Astrocytes maintain homeostasis through various mechanisms, including EV release. The effect of these EVs on mitigating neurotoxicity in HBECs has not been investigated. This study assesses the impact of astrocyte-derived EVs on global transcriptome changes, cell proliferation, cytotoxicity, oxidative DNA damage, and mitochondrial morphology in HBECs exposed to the neurotoxic reagent Na2Cr2O7. Exposure to Na2Cr2O7 for 5 and 16 h induced oxidative DNA damage, measured by an increase in genomic 8OHdG, while the EVs reduced the accumulation of the adduct. A neurotoxic environment caused a non-statistically significant upregulation of the DNA repair enzyme OGG1 while the addition of astrocyte-derived EVs was associated with the same level of expression. EVs caused increased cell proliferation and reduced cytotoxicity in Na2Cr2O7-treated cells. Mitochondrial dysfunction associated with a reduced copy number and circular morphology induced by neurotoxic exposure was not reversed by astrocyte-derived EVs. High-throughput RNA sequencing revealed that exposure to Na2Cr2O7 suppressed immune response genes. The addition of astrocyte-derived EVs resulted in the dysregulation of long noncoding RNAs impacting genes associated with brain development and angiogenesis. These findings reveal the positive impact of astrocytes-derived EVs in mitigating neurotoxicity and as potential therapeutic avenues for neurodegenerative diseases.

Extracellular vesicles-derived microRNAs expression as biomarkers for neurological radiation injury: Risk assessment for space exploration.

Space missions pose threats to the health of the astronauts due to long-term exposure to galactic cosmic rays and solar particle events comprised predominantly of medium to high energy protons, energetic helium ions, and energetic high atomic number particles (HZEs). While the tissue-specific effects of radiation have been studied extensively, the changes in exosomal miRNA expression levels in response to acute radiation exposure have not been assessed. Extracellular vesicles (EVs) originate from the host cells and contain nucleic acid and proteins that can modify the physiology of the receiving cells via the transfer of genomic, proteomic, and lipids cargo. Detection and analysis of miRNA cargo of circulating EVs is an emerging method for non-invasive diagnosis and monitoring of neurological disorders. This study characterizes the EV-derived miRNA expression profiles of human astrocytes to identify those that are altered after treatment with 3 Gy proton radiation as biomarkers of neurological radiation injury. The relationship between radiation and miRNA extracellular vesicles expression levels was investigated in human astrocytes after treatment with 3 Gy proton radiation at Willis-Knighton Cancer Center. Microarray analysis was performed using miRNA from the EVs enriched fraction in the cell culture medium collected from sham-control and radiation-treated cells. The exosomal levels of 13 miRNAs were significantly (FDR p < 0.05) down-regulated after exposure to high-energy radiation. The computational analysis identified hsa-miR-762, hsa-let-7c-5p, and has-let-7b-5p regulate the highest number of genes being associated with cognitive, mental, and motor delay. These miRNAs target the same subset of genes (Amd1, CCNF, COX6B, PLXND1) that are associated with epileptic encephalopathy; frontotemporal dementia; mitochondrial complex iv deficiency, and a rare neurological condition (Moebius syndrome) respectively. GO enrichment analysis of the biological processes identified overrepresentation in mRNA polyadenylation and regulation of glutamine and long fatty acids transport. Gene expression analysis confirmed the upregulation of the glutamine synthetase after irradiation. Significant fold enrichment of GO l-glutamine transmembrane transporter activity was identified in the molecular function category as well indicating exosome-mediated regulation of this important pathway after proton radiation exposure.

Identification of microRNA-mRNA regulatory network associated with oxidative DNA damage in human astrocytes.

The high lipid content of the brain, coupled with its heavy oxygen dependence and relatively weak antioxidant system, makes it highly susceptible to oxidative DNA damage that contributes to neurodegeneration. This study is aimed at identifying specific ROS-responsive miRNAs that modulate the expression and activity of the DNA repair proteins in human astrocytes, which could serve as potential biomarkers and lead to the development of targeted therapeutic strategies for neurological diseases. Oxidative DNA damage was established after treatment of human astrocytes with 10µM sodium dichromate for 16 h. Comet assay analysis indicated a significant increase in oxidized guanine lesions. RT-qPCR and ELISA assays confirmed that sodium dichromate reduced the mRNA and protein expression levels of the human base-excision repair enzyme, 8-deoxyguanosine DNA glycosylase 1 (hOGG1). Small RNAseq data were generated on an Ion Torrent™ system and the differentially expressed miRNAs were identified using Partek Flow® software. The biologically significant miRNAs were selected using miRNet 2.0. Oxidative-stress-induced DNA damage was associated with a significant decrease in miRNA expression: 231 downregulated miRNAs and 2 upregulated miRNAs (p < 0.05; >2-fold). In addition to identifying multiple miRNA-mRNA pairs involved in DNA repair processes, this study uncovered a novel miRNA-mRNA pair interaction: miR-1248:OGG1. Inhibition of miR-1248 via the transfection of its inhibitor restored the expression levels of hOGG1. Therefore, targeting the identified microRNA candidates could ameliorate the nuclear DNA damage caused by the brain's exposure to mutagens, reduce the incidence and improve the treatment of cancer and neurodegenerative disorders.

ExoPRIME: Solid-phase immunoisolation and OMICS analysis of surface-marker-specific exosomal subpopulations.

Exosomes encapsulate genomic and proteomic biomarkers for non-invasive diagnosis and disease monitoring. However, exosome surface-markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one-step exosome sampling technology, ExoPRIME, for selective capture of CD63+ exosome subpopulations using an immune-affinity protocol. Microneedles (300µm × 30 mm), functionalized with anti-CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte-derived exosome suspension. The probe's capture efficiency and specificity were validated using FluoroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 0C in enriched exosomal suspension (23 × 10 6 exosomes per probe) vis-à-vis 2 h at 4 0 C (12 × 10 6) and 16 h at 22 0C (3 × 10 6) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re-use of minimal sample volumes (≤200 µL), can be multiplexed in arrays, and integrated into a lab-on-a-chip platform to achieve parallel, high-throughput isolation of different exosome classes in a semi-automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room-temperature, picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care.

Photon versus proton neurotoxicity: Impact on mitochondrial function and 8-OHdG base-excision repair mechanism in human astrocytes.

This study assesses and compares the neurotoxic effects of proton and photon radiation on mitochondrial function and DNA repair capabilities of human astrocytes. Human astrocytes received either proton (0.5 Gy and 3 Gy), photon (0.5 Gy and 3 Gy), or sham-radiation treatment. The mRNA expression level of the DNA repair protein OGG1 was determined via RT-qPCR. The levels of 8-OHdG in the cell media were measured via ELISA. Real-time kinetic analysis of extracellular oxygen consumption rates was performed to assess mitochondrial function. Radiation-induced changes in mitochondrial mass and oxidative activity were assessed using fluorescent imaging with MitoTracker™ Green FM and MitoTracker™ Orange CM-H2TMRos dyes respectively. PCR was used to quantify the alteration in the mitochondrial DNA content, measured as the mitochondrial to nuclear DNA ratio. A significant increase in mitochondrial mass and levels of reactive oxygen species was observed after radiation treatment. Additionally, real-time PCR analysis indicated a significant depletion of mitochondrial DNA content in the irradiated cells when compared to the control. This was accompanied by a decreased gene expression of the DNA base-excision repair protein OGG1 and reduced clearance of 8-OHdG adducts from the genome. Photon radiation treatment was associated with a more detrimental cellular impact when compared to the same dose of proton radiation. These results are indicative of a radiation-induced dose-dependent decrease in mitochondrial function, an increase in senescence and astrogliosis, and impairment of the DNA repair capabilities in healthy glial cells. Photon irradiation was associated with a more significant disruption in mitochondrial function and base-excision repair mechanisms in vitro in comparison to proton treatment.

Calorimetric sandwich-type immunosensor for quantification of TNF-α.

We report a lab-on-a-chip immunosesnor for quantification of the inflammatory cytokine TNF-α with picomolar sensitivity. The feasibility of the technology was demonstrated via accurate measurement of the concentration of TNF-α in astrocytes cell culture media. The immunoassay was performed in a microfluidic device with an integrated antimony/bismuth thermopile sensor and had a limit of detection of 14 pg mL-1. The device was fabricated using rapid prototyping xurography technique and consisted of two inlets and single outlet. Anti-TNF-α monoclonal antibody was used to capture the analyte while the detection was performed using glucose oxidase-conjugated secondary antibody. Glucose (55 mM) was injected through a sample loop into the fluid flowing within the microfluidic device. A nanovolt meter connected to the thermoelectric sensor recorded the voltage change caused by the enzymatic reaction. Computer simulations using COMSOL Multiphysics were performed to analyze the effect of fluid velocity on the concentration of glucose within the reaction zone. A standard calibration curve was created using serial dilutions of synthetic TNF-α (0-2000 pg mL-1) by plotting the area under the curve of the signal versus the concentration of the analyte. The efficacy of the device was evaluated by quantifying TNF-α in the cell culture medium of lipopolysaccharide stimulated and non-stimulated astrocytes. The results demonstrated high accuracy of the calorimetric immunoassay when compared with gold standard commercial ELISA microplate reader. The immunosensor offers excellent reproducibility, accuracy, and versatility in the choice of the detection enzyme.

Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 µm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

Thermoelectric method for sequencing DNA.

This study describes a novel, thermoelectric method for DNA sequencing in a microfluidic device. The method measures the heat released when DNA polymerase inserts a deoxyribonucleoside triphosphate into a primed DNA template. The study describes the principle of operation of a laminar flow microfluidic chip with a reaction zone that contains DNA template/primer complex immobilized to the inner surface of the device's lower channel wall. A thin-film thermopile attached to the external surface of the lower channel wall measures the dynamic change in temperature that results when Klenow polymerase inserts a deoxyribonucleoside triphosphate into the DNA template. The intrinsic rejection of common-mode thermal signals by the thermopile in combination with hydrodynamic focused flow allows for the measurement of temperature changes on the order of 10(-4) K without control of ambient temperature. To demonstrate the method, we report the sequencing of a model oligonucleotide containing 12 bases. Results demonstrate that it is feasible to sequence DNA by measuring the heat released during nucleotide incorporation. This thermoelectric method for sequencing DNA may offer a novel new method of DNA sequencing for personalized medicine applications.