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.

Recent Advances in the Fabrication and Application of Screen-Printed Electrochemical (Bio)Sensors Based on Carbon Materials for Biomedical, Agri-Food and Environmental Analyses.

This review describes recent advances in the fabrication of electrochemical (bio)sensors based on screen-printing technology involving carbon materials and their application in biomedical, agri-food and environmental analyses. It will focus on the various strategies employed in the fabrication of screen-printed (bio)sensors, together with their performance characteristics; the application of these devices for the measurement of selected naturally occurring biomolecules, environmental pollutants and toxins will be discussed.

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

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.

Use of whole blood for analysis of disease-associated biomarkers.

Analyses for diagnosis and monitoring of pathological conditions often rely on blood samples, partly due to relative ease of collection. However, many interfering substances largely preclude the use of whole blood itself, necessitating separation of plasma or serum. We present a feasibility study demonstrating potential use of fresh or frozen whole blood to detect soluble biomarkers using an enzyme-linked immunosorbent assay (ELISA)-based method. Good correlation between levels of soluble CD25 in plasma and whole blood of healthy individuals or Alzheimer's patients was established. These results provide a basis for development of a novel biosensor approach for disease-associated biomarker detection in whole blood.

Development of an amperometric screen-printed galactose biosensor for serum analysis.

The development of a disposable amperometric biosensor for the measurement of circulating galactose in serum is described. The biosensor comprises a screen-printed carbon electrode (SPCE), incorporating the electrocatalyst cobalt phthalocyanine (CoPC), which is covered by a permselective cellulose acetate (CA) membrane and a layer of immobilized galactose oxidase (GALOX). The optimal response of the biosensor, designated as GALOX-CA-CoPC-SPCE, was obtained by systematically examining the effects of enzyme loading, temperature, pH, and buffer strength. The optimal performance of the biosensor occurred with 2U of GALOX, at 35°C, using 50mM phosphate buffer solution (pH 7.0). The sensitivity was 7.00µAmM(-1)cm(-2) and the linear range from 0.1 to 25mM with a calculated limit of detection (LOD) of 0.02mM; this concentration range and LOD are appropriate to diagnose galactosemia, i.e., concentrations >1.1mM in infants. When the biosensor was used in conjunction with amperometry in stirred solution for the analysis of serum, the precision values obtained on unspiked (endogenous level of 0.153mM) and spiked serum (1mM added) (n=6) were 1.10% and 0.11%, respectively, with a calculated recovery of 99.9%.

Preparation of screen-printed electrochemical immunosensors for estradiol, and their application in biological fluids.

The method of fabrication of a prototype electrochemical immunosensor for estradiol (E2) is described. Methodologies are also given for colorimetric assays, which can be used to verify and optimize reagent performance, prior to their use in the electrochemical immunoassay: these include an E2 ELISA and a colorimetric assay performed on the immunosensor surface. The electrochemical immunosensor system uses screen-printed carbon electrodes (SPCEs) upon which antibody against E2 is immobilized. Antibodies (rabbit anti-mouse IgG, then monoclonal mouse anti-E2) are immobilized by passive adsorption onto the working electrode surface. A competitive immunoassay is then performed using an alkaline-phosphatase-labeled E2 conjugate. Electrochemical measurements are performed using differential pulse voltammetry (DPV) to detect the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current vs. E2 concentration shows a measurable range of 25-500 pg/mL with a detection limit of 50 pg/mL. The immunosensor can be applied to the determination of E2 in spiked serum, following an extraction step with diethyl ether.