Amperometric Screen-Printed Galactose Biosensor for Cell Toxicity Applications

© 2016, Copyright © Taylor & Francis Group, LLC. ABSTRACT: This paper reports the development and application of a biosensor for the amperometric determination of galactose in the presence of human hepatocellular carcinoma cells with and without a hepatotoxic agent. The biosensor was fabricated by drop-coating 1.5% cellulose acetate on a 3×3mm screen-printed carbon electrode followed by depositing 2 U of galactose oxidase. The electrodes dimensions were reduced to 3×0.5mm before measurements. Hepatocellular carcinoma cells were utilized for in vitro toxicity testing by evaluating the effect of paracetamol on galactose uptake. The amperometric responses to galactose indicated that the inhibition of uptake was directly proportional to the concentration of paracetamol following 24h of exposure to the hepatocellular carcinoma cells. These results demonstrate that the fabricated biosensor may be used for the real-time monitoring of cell metabolism and toxicity

Development of a disposable screen-printed amperometric biosensor based on glutamate dehydrogenase, for the determination of glutamate in clinical and food applications

A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola’s Blue (MB) has been investigated as the base transducer for a glutamate biosensor. The sandwich biosensor was fabricated by firstly depositing a chitosan (CHIT) layer onto the surface of the transducer (MB-SPCE), followed by glutamate dehydrogenase (GLDH): this device is designated GLDH-CHIT-MB-SPCE. NAD+ was added to buffer solutions prior to the measurement of glutamate. This biosensor was used in conjunction with amperometry in stirred solution at an applied potential of +0.1 V (vs. Ag/AgCl). Optimum conditions for the analysis of glutamate were found to be as follows: temperature, 35°C; buffer, pH 7; ionic strength, 75 mM; NAD+, 4 mM; CHIT 0.05% in 0.05 M HCl; GLDH, 30 U. The linear range of the biosensor was found to be 12.5 µM to 150 µM, the calculated limit of detection (based on three times signal to noise ratio) was 1.5 µM and the sensitivity was 0.44 nA/µM. The proposed biosensor was used to measure glutamate in serum before and after fortification with glutamate. The endogenous concentration of glutamate was found to be 1.68 mM and the coefficient of variation (CV) was 4.1%. The serum was then fortified with 2 mM of glutamate, and the resulting mean recovery was 96% with a CV of 3.3% (n = 6). An unfiltered beef OXO cube was analysed for monosodium glutamate (MSG) content. The endogenous content of MSG was 124.80 mg/g with a CV of 8.06%. The OXO cube solution was fortified with 0.935g (100 mM) of glutamate, the resulting mean recovery was 91% with a CV of 6.40 %

Fabrication and evaluation of a micro(bio)sensor array chip for multiple parallel measurements of important cell biomarkers

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This report describes the design and development of an integrated electrochemical cell culture monitoring system, based on enzyme-biosensors and chemical sensors, for monitoring indicators of mammalian cell metabolic status. MEMS technology was used to fabricate a microwell-format silicon platform including a thermometer, onto which chemical sensors (pH, O2) and screen-printed biosensors (glucose, lactate), were grafted/deposited. Microwells were formed over the fabricated sensors to give 5-well sensor strips which were interfaced with a multipotentiostat via a bespoke connector box interface. The operation of each sensor/biosensor type was examined individually, and examples of operating devices in five microwells in parallel, in either potentiometric (pH sensing) or amperometric (glucose biosensing) mode are shown. The performance characteristics of the sensors/biosensors indicate that the system could readily be applied to cell culture/toxicity studies

A novel in vitro 3D model of the human bone marrow to bridge the gap between in vitro and in vivo genotoxicity testing

The regulatory 2D in vitro micronucleus (MN) assay is part of a battery of tests, used to test for genotoxicity of new and existing compounds before they are assessed in vivo (ICH S2). The 2D MN assay consists of a monolayer of cells, whereas the in vivo bone marrow (BM) setting comprises a multicellular environment within a three-dimensional extracellular matrix. Although the in vitro MN assay follows a robust protocol set out by the Organisation for Economic Co-operation and Development (OECD) to comply with regulatory bodies, some compounds have been identified as negative genotoxicants within the in vitro MN assay but marginally positive when assessed in vivo. The glucocorticoids, which are weakly positive in vivo, have generally been suggested to pose no long-term carcinogenic risk; however, for novel compounds of unknown activity, improved prediction of genotoxicity is imperative. To help address this observation, we describe a novel 3D in vitro assay which aims to replicate the results seen within the in vivo BM microenvironment. AlgiMatrix scaffolds were optimized for seeding with HS-5 human BM stromal cells as a BM microenvironment, to which the human lymphoblast cell line TK6 was added. An MN assay was performed aligning with the 2D regulatory assay protocol. Utilizing this novel 3D in vitro model of the BM, known genotoxicants (mitomycin C, etoposide, and paclitaxel), a negative control (caffeine), and in vivo positive glucocorticoids (dexamethasone and prednisolone) were investigated for the induction of MN. It was found, in agreement with historical in vivo data, that the model could accurately predict the in vivo outcome of the glucocorticoids, unlike the regulatory 2D in vitro MN assay. These preliminary results suggest our 3D MN assay may better predict the outcome of in vivo MN tests, compared with the standard 2D assay

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism—the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1—a gene know to regulate skeletal development—is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization

Screen-printed carbon based biosensors and their applications in agri-food safety

This review focuses on the ways in which screen-printed carbon electrodes have been tailored with different biorecognition elements, including enzymes, antibodies, and aptamers, often with other modifiers, such as mediators and nanoparticles, to produce electrochemical biosensors for a variety of analytes of importance in agri-food safety. Emphasis is placed on the strategies of biosensor fabrication and the performance characteristics of the devices. As well as biosensors for a range of analytes in different agri-food matrices, we have also included reports on novel devices that have potential in agri-food safety but as yet have not been applied in this area

Delivering a sustainable trauma management training programme tailored for low-resource settings in East, Central and Southern African countries using a cascading course model

Background: Injuries cause five million deaths and 279 Disability Adjusted Life Years (DALYS) each year worldwide. The COSECSA Oxford Orthopaedic Link (COOL) is a multi-country partnership programme that has delivered training in trauma management to nine sub-Saharan countries across a wide-cadre of health-workers using a model of "primary" courses delivered by UK instructors, followed by "cascading" courses led by local faculty. This study examines the impact on knowledge and clinical confidence among health-workers, and compares the performance of "cascading" and "primary" courses delivered in low-resource settings. Methods: Data was collated from 1030 candidates (119 Clinical Officers, 540 Doctors, 260 Nurses and 111 Medical Students) trained over 28 courses (9 "primary" and 19 "cascading" courses) in nine sub-Saharan countries between 2012 and 2013. Knowledge and clinical confidence of candidates were assessed using pre- and post-course MCQs and confidence matrix rating of clinical scenarios. Changes were measured in relation to co-variants of gender, job roles and primary versus cascading courses. Multivariate regression modelling and cost analysis was performed to examine the impact of primary versus cascading courses on candidates' performance. Findings: There was a significant improvement in knowledge (58% to 77%, p < 0.05) and clinical confidence (68% to 90%, p < 0.05) post-course. "Non-doctors" demonstrated a greater improvement in knowledge (22%) and confidence (24%) following the course (p < 0.05). The degree of improvement of MCQ scores differed significantly, with the cascading courses (21%) outperforming primary courses (15%) (p < 0.002). This is further supported by multivariate regression modelling where cascading courses are a strong predictor for improvement in MCQ scores (Coef = 4.83, p < 0.05). Interpretation: Trauma management training of health-workers plays a pivotal role in tackling the ever-growing trauma burden in Africa. Our study suggests cascading PTC courses may be an effective model in delivering trauma training in low-resource settings, however further studies are required to determine its efficacy in improving clinical competence and retention of knowledge and skills in the long term