Thermoelectric ELISA for quantification of 8OHdG in a microfluidic device

This research demonstrates the feasibility of a novel method for performing thermoelectric enzyme-linked immunosorbent assay (ELISA) in a microfluidic device. The feasibility of the thermoelectric ELISA is demonstrated by measuring the concentration of 8-hydroxy 2-deoxyguanosine (8OHdG) in urine samples from amyloid precursor protein (APP) transgenic mice. The detection method is based on formation of a complex between 8OHdG and anti-8OHdG capture antibody conjugated to biotin. The complex is immobilized over the measuring junctions of a thermopile via biotin streptavidin interaction. The concentration of the analyte is determined by using enzyme linked secondary IgG antibody specific to the primary one. The concentration of 8OHdG is determined by the initiation of an enzymatic reaction between glucose and glucose oxidase that is conjugated to the secondary IgG antibody. The heat released by the reaction of glucose and glucose oxidase is measured using an antimony-bismuth thermopile integrated in a microfluidic device. The amount of heat detected by the sensor is inversely proportional to the concentration of 8OHdG. A standard calibration curve using known concentrations of synthetic 8OHdG is generated and used to determine the concentration of the oxidized guanine in mouse urine samples

Lab-on-a-Chip Calorimetric Immunosensor: Computational Analysis and Feasibility Study

Gergana G. Nestorova is an Assistant Professor in the School of Biological Sciences at Louisiana Tech University. Saif M. I. Bari is a graduate student in Micro and Nanoscale Systems Engineering at Louisiana Tech University. To view the abstract for their presentation Lab-on-a-Chip Calorimetric Immunosensor: Computational Analysis and Feasibility Study click on the blue download button

Effect of High-Energy Radiation on the Formation of 8- Hydroxy-Deoxyguanosine

The abstract for this presentation can be downloaded by clicking on the blue download button

01. ExoSense: a microprobe-based method for single-step isolation and genetic of exosomes

The goal of this study is to develop a new method for non-invasive and selective isolation of exosomes from cell media. Exosomes, a type of extracellular vesicles, contain proteins and RNA biomarkers for the diagnosis of diseases. The core of this technology is a stainless-steel microprobe (300μm × 30mm) functionalized with anti-CD63 antibodies that specifically capture pure exosomal subpopulations. This method provides several advantages over commercial exosome-purification technologies. These include increased selectivity, purification of an antigen-specific subpopulation of exosomes, and direct integration of the microprobe with genetic analysis instruments. Experiments were performed to assess the efficiency of the functionalization of the probe as well as the number of captured exosomes, exosomal protein, and RNAs per probe. Scanning electron imaging was used to visualize the polyelectrolyte coverage while fluorescent imaging was applied to determine the efficiency of the chemical bond of NH2-conjugated biotin to the COOH group of polyacrylic acid. The captured exosomes were quantified by measuring the activity of acetylcholinesterase (AChE). The BCA protein assay was utilized to quantify the amount of protein captured per microprobe while Agilent Bioanalyzer 2100 was used to assess the quantity and quality of the exosomal RNA. Our results indicate that the microprobe-based technology isolates CD63-positive exosomal subpopulation (23×106 exosomes/probe) from astrocytes derived cell media after 16 hours of incubation. The exosomal fraction is enriched in small RNAs. Future work will focus on the assessment of blocking reagents (BSA, PEG) to reduce the binding of the cell-media-derived protein to the surface of the probe

Identification of Novel MicroRNAs that Regulate OGG1 Mediated DNA Repair

Kristen H. Hutson and Kaitlynn M. Willis are students in the School of Biological Sciences at Louisiana Tech University. Gergana G. Nestorova is an Assistant Professor in the School of Biological Sciences at Louisiana Tech University

Microprobe-based Platform for Rapid Immunocapture and Genetic Analysis of Exosomes

Dr. Gergana G. Nestorova is an Assistant Professor in the School of Biological Sciences at Louisiana Tech University. Chukwumaobim D. Nwokwu is a doctoral student in Molecular Science and Nanotechnology at Louisiana Tech University. Saif Mohammad Ishraq Bari is a doctoral student in Micro and Nanoscale Systems Engineering at Louisiana Tech University. The abstract for this presentation can be downloaded by clicking on the blue download button

Effect of S on Mitochondrial DNA Copy Number

Kaitlynn M. Willis and Kristen H. Hutson are students in the School of Biological Sciences at Louisiana Tech University. Gergana G. Nestorova is an Assistant Professor in the School of Biological Sciences at Louisiana Tech University

Identification of miRNA-OGG1 mRNA Interactions: Small RNA Sequencing and Immunoprecipitation Analysis

The abstract for this presentation can be downloaded by clicking on the blue download button

Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality

BACKGROUND: Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse. METHODS AND FINDINGS: We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes. CONCLUSIONS: This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment

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