Development of whole cell-based biosensor in 3D environment for nanoparticles cytotoxicity detection

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, three dimensional environments for human cells were developed and tested using three different types of hydrogels including transglutaminase-crosslinked gelatin, collagen type I and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, urea and albumin analysis indicated that the created system allows the immortalized liver cell line (HepG2) to re-establish normal hepatocyte-like properties which are not observed under the conditions of conventional cell cultures. This study presents a reproducible technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates higher cytotoxic effects when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that cell environment has significant influence on cell sensitivity and that liver spheroids are useful and novel tools to examine NP dosing effect even at the level of in vitro studies. Moreover, the created cell-based biosensor responds optimally to bioactive analytes, has a fast response time, offers label-free experimentation and simple procedures. Besides, the developed NF-κB_HepG2 cells enable detect cytotoxicity of variety nanoparticles with different chemical composition, size, tendency of agglomeration or cytotoxic potential and other stimuli which activate NF-κB signaling (e.g. heavy metals), in very early stage, before cell death, in short time even after 2 h time exposure as opposed to widely used commercially available cell-based assays like MTT, XTT Alamar Blue or LDH. Successful modification of HepG2 cells resulted in generation of novel NF-κB_HepG2 sensor cells with extracellular reporter protein (human secreted alkaline phosphatase) and its application in cytotoxicity of nanomaterials detection has not been investigated before. The developed NF-κB_hepG2 cells may also provide a useful tool to study distinct molecular mechanisms of inflammation and cellular stress using low exposure concentrations of cell irritants. The presented sensor based on human cells in three dimensional milieu provides a novel application for nanoparticles screening that joins the complex in vitro model imitating living tissue with high throughput analytical methods. This system can be applied to a wide diversity of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions

DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex DSBs, but in 40% of the isolated DSBs the recruitment was delayed approximately 5 min. Using base excision repair (BER) inhibitors we demonstrate that some simple DSBs are generated by enzymatic processing of base damage, while BER did not affect the complex DSBs. The results show that DSB processing and repair kinetics are dependent on the complexity of the breaks and can be different even for the same clastogenic agent

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL(-1) and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL(-1) by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied. (C) 2016 Elsevier B.V. All rights reserved

Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes

The rapid and sensitive detection of aflatoxin M-1 (AFM(1)) in milk by using surface plasmon resonance (SPR) biosensor is reported. This low molecular weight mycotoxin is analyzed using an indirect competitive immunoassay that is amplified by secondary antibodies conjugated with Au nanoparticles. In order to prevent fouling on the sensor surface by the constituents present in analyzed milk samples, an interface with poly(2-hydroxyethyl methacrylate) p(HEMA) brush was employed. The study presents a comparison of performance characteristics of p(HEMA)-based sensor with a regularly used polyethylene glycol -based architecture relying on mixed thiol self-assembled monolayer. Both sensors are characterized in terms of surface mass density of immobilized AFM(1) conjugate as well as affinity bound primary and secondary antibodies. The efficiency of the amplification strategy based on Au nanoparticle is discussed. The biosensor allowed for highly sensitive detection of AFM(1) in milk with a limit of detection (LOD) as low as 18 pg mL(-1) with the analysis time of 55 min. (C) 2016 Elsevier B.V. All rights reserved