Label-Free, Highly Sensitive Electrochemical Aptasensors Using Polymer-Modified Reduced Graphene Oxide for Cardiac Biomarker Detection

Acute myocardial infarction (AMI), also recognized as a ???heart attack,??? is one leading cause of death globally, and cardiac myoglobin (cMb), an important cardiac biomarker, is used for the early assessment of AMI. This paper presents an ultrasensitive, label-free electrochemical aptamer-based sensor (aptasensor) for cMb detection using polyethylenimine (PEI)-functionalized reduced graphene oxide (PEI???rGO) thin films. PEI, a cationic polymer, was used as a reducing agent for graphene oxide (GO), providing highly positive charges on the rGO surface and allowing direct immobilization of negatively charged single-strand DNA aptamers against cMb via electrostatic interaction without any linker or coupling chemistry. The presence of cMb was detected on Mb aptamer-modified electrodes using differential pulse voltammetry via measuring the current change due to the direct electron transfer between the electrodes and cMb proteins (Fe3+/Fe2+). The limits of detection were 0.97 pg mL???1 (phosphate-buffered saline) and 2.1 pg mL???1 (10-fold-diluted human serum), with a linear behavior with logarithmic cMb concentration. The specificity and reproducibility of the aptasensors were also examined. This electrochemical aptasensor using polymer-modified rGO shows potential for the early assessment of cMb in point-of-care testing applications

MICRO-PHYSIOLOGICAL MODELS TO MIMIC MUCOSAL BARRIER COMPLEXITY OF THE HUMAN INTESTINE IN VITRO

The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material (i.e. functional mucins) and platforms to integrate a relevant thickness of mucus layer with an epithelium under physiological conditions. Here we report our progress on developing humanized micro-physiological models of the intestines in static and dynamic settings by using natural mucins derived from porcine small intestines (PSI). To overcome limited availability of mucus, we developed a simple and scalable method for mucus extraction by directly solubilizing the luminal mucus layer from PSI that is readily accessible.The mucus barrier was successfully integrated with human epithelial cell layer (HT-29) co-cultured with immune cells (THP-1), which allowed the studies of bi-directional crosstalk between luminal content and tissue immune cells through a physiologically relevant mucosal interface. The applied mucus barrier did not cause any cytotoxic or immunogenic effects to human intestinal and immune cells. As expected, mucus prevents the transmigration of probiotic bacteria VSL#3. In the absence of mucus, these bacteria caused epithelial damage, immune cell differentiation and induced production of pro-inflammatory cytokines IL-8 and TNF-α. The most intriguing result from these studies was that mucus increased the transmigration of pathogenic Salmonella. Breach of the mucosal barrier by Salmonella induced production of IL-8 and TNF-α. Taking bioengineering approaches, we have developed mucosal barrier models of intestines under static (transwells) and dynamic conditions (microfluidics). Established models represent cellular and extracellular complexities in a controlled and accessible manner. We envision that in vitro mucosal barrier models will serve as an enabling tool for understanding basic biology and disease progression in the intestines

The role of IGF-1 in the development and progression of colorectal cancer

Colorectal Cancer (CRC) is one of the most prevalent cancers among men and women in the world. Its presence is increasingly being felt in people who east a western diet. A sedentary life, lack of physical activity, and excessive consumption of red meat are some of the major risk factors associated with the disease. Furthermore, increasing correlations are demonstrating that diabetic and obese patients are at a higher risk of developing colorectal cancer. Of increasing interest is the presence of growth factors present in these individuals. One such growth factor, which is being crucially studied for the purposes of treatment and prevention, is insulin-like growth factor-1 (IGF-1.) IGF-1 is part of the IGF axis, an endocrine axis, composed of IGF-1 receptor (IGF-1R), numerous IGF binding proteins (IGFBPs), and insulin. Many studies have highlighted the high levels of IGF-1 in the blood of patients with CRC. However, currently IGF-1 is only regarded as a biomarker for CRC. The role that a dysregulated IGF axis plays in the development and progression of CRC is largely unknown. This review of the current literature has underlined major risk factors of colorectal cancers which are able to exert their unwanted effect through their impact on the IGF axis. These factors include obesity, diabetes, radiotherapy, and oxidative stress. The understanding of the dysregulation of IGF axis’ pathways in colorectal cancer is crucial for the development of new treatment options for the disease in the future. In this paper the known mechanisms through which IGF-1s binding to IGF-1R causes unnecessary cellular proliferation in colorectal cancer patients is discussed. IGF-1R is a dimer with a tyrosine kinase domain on the intracellular side. Ligand binding to this receptor causes autophosphrylation and the activation of the Phosphoinositide 3-kinase/Protein Kinase B/Wnt (PI3K/Akt/Wnt) pathway. The PI3K/Akt/Wnt pathway is known to cause cellular proliferation and, in excess, metastasis. The potential for the IGF axis to cause metastasis will also be discussed in more direct terms. Levels of IGF-1R and IGF-1 are positively correlated with different stages of metastasis. Furthermore, in vitro and in vivo studies have demonstrated that IGF-1 has the ability to increase lymphatic vessel density. The potential for changes in the treatment of patients with colorectal cancer and prevention of the disease in those who are at risk of it is also discussed. Currently the main therapies through which the IGF axis is targeted are chemotherapy and immunotherapy. However, these therapies can cause a lot of damage to the body as they are not specific to the cancer cells. A new theory known as the cancer stem cell theory is readily being used to design treatment options for the future. According to this theory, cancer stem cells are present in small amounts in everyone and are responsible for the relapse of cancer after radiation or chemotherapy. These cells are increasingly being targeted for drug design as they provide specificity which would, hypothetically, not produce as many side effects. Another mechanism, which provides specificity and is gaining popularity in treating CRC, involves the use of microRNAs. microRNAs are small RNAs which can bind to mRNAs of major genes and prevent their translation. miRNAs which suppress the genes of the IGF axis can be administered to the patient to specifically address the problem. Numerous such miRNAs have been discovered and their roles were discussed in further detail. Lastly, the ability of some bioactive compounds to perturb the IGF axis in CRC is also discussed. These compounds are mainly found in some of the fruits and vegetables, and spices like curcumin. Vitamin D is another dietary compound which has the potential to prevent CRC in the susceptible population. However the extent to which small amounts of these compounds can prevent CRC is largely indeterminate and there is a need to conduct studies on the compounds’ dosing regimen. The review of the literature on colorectal cancer has discussed some of the latest strategies in tackling the disease as well as providing mechanistic insights into IGF axis’ dysregulation in CRC

ESTIMATING THE EFFECTS OF BLASTING VIBRATIONS ON THE HIGH-WALL STABILITY

The stability of the high-walls is one of the major concerns for open pit mines. Among the various factors affecting the stability of high-walls, blast vibrations can be an important one. In general, worldwide the established respective government regulations and industry standards are used as guidance to determine the maximum recommended levels of the peak particle velocity and frequency from the blast to avoid any effects on the structures around the mining project. However, most of the regulations are meant for buildings or houses and do not concern high-walls. This thesis investigates the response of high-walls under the effects of vibrations from mine blasting. In this research, the relationship between the high-wall response, the geometry of the slope, the frequency and the amplitude, of the ground vibration produced by blasting, is explored using numerical models in 3DEC. The numerical models were calibrated initially with data collected using seismographs installed in a surface mine operation and recording vibrations produced by an underground mine drill and blast operation. Once the calibration was accomplished, a parametric study was developed to explore the relationships between various parameters under study and its impact on the stability of high-walls

Normal stress anisotropy and marginal stability in athermal elastic networks

Hydrogels of semiflexible biopolymers such as collagen have been shown to contract axially under shear strain, in contrast to the axial dilation observed for most elastic materials. Recent work has shown that this behavior can be understood in terms of the porous, two-component nature and consequent time-dependent compressibility of hydrogels. The apparent normal stress measured by a torsional rheometer reflects only the tensile contribution of the axial component $\sigma_{zz}$ on long (compressible) timescales, crossing over to the first normal stress difference, $N_1 = \sigma_{xx}-\sigma_{zz}$ at short (incompressible) times. While the behavior of $N_1$ is well understood for isotropic viscoelastic materials undergoing affine shear deformation, biopolymer networks are often anisotropic and deform nonaffinely. Here, we numerically study the normal stresses that arise under shear in subisostatic, athermal semiflexible polymer networks. We show that such systems exhibit strong deviations from affine behavior and that these anomalies are controlled by a rigidity transition as a function of strain

Flexible electrical aptasensor using dielectrophoretic assembly of graphene oxide and its subsequent reduction for cardiac biomarker detection

Cardiac troponin T (cTnT) is considered a clinical standard for its high specificity and sensitivity when diagnosing acute myocardial infarction; however, most studies on the electrical sensors of cardiac troponin biomarkers have focused on cTnI rather than cTnT. This study presents label-free, low-cost, transparent, and flexible aptamer-based immunosensors for the electrical detection of cTnT using reduced graphene oxide (rGO) sheets. GO was first deposited by AC dielectrophoresis between two predefined source and drain electrodes on a 3-aminopropyltriethoxylsilane-modified polyethylene terephthalate substrate. The GO was then reduced using hydrazine vapour without damaging the substrate, resulting in uniform, controlled, and stable deposition of rGO sheets, and demonstrating more stability than those directly deposited by dielectrophoresis. Amine-modified single-strand DNA aptamers against cTnT were immobilized onto the rGO channels. The relative resistance change of this sensor owing to the attachment of cTnT was quantified as the cTnT concentration decreased from 10???ng/mL to 1???pg/mL in phosphate buffered saline (PBS) and 10-fold diluted human serum in PBS, with the limits of detection being 1.2???pg/mL and 1.7???pg/mL, respectively, which is sufficiently sensitive for clinical applications. High-yield and rapid fabrication of the present rGO sensors will have significant influences on scaled-up fabrication of graphene-based sensors