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High level Integrated SEnsor for NanoToxicity Screening

Periodic Reporting for period 2 - HISENTS (High level Integrated SEnsor for NanoToxicity Screening)

Reporting period: 2017-10-01 to 2019-03-31

HISENTS goal was to develop a smart multimodule screening technology for nanosafety assessment with mechanistic functionality. This will provide "how" and "why" answers to the fundamental processes of human response to nanomaterial (NM) toxicity using toxicity pathway analysis, toxicogenomics, and novel endpoints. For this purpose an innovative miniaturised high throughput screening (HTS) tool has been developed with the capability of reliably analysing bio-nano-interactions within a platform of increasing complexity of biological organisation. HISENTS has also aligned this experimental platform with a comprehensive physiologically based pharmacokinetic (PBPK) type model to aid in the platform design and to contribute to the pathway and mechanistic analysis. The PBPK model together with the instrumentation development is integrated into one multimodular platform screen.

HISENTS is important to society as:
1. It leads to a step increase in the speed of screening nanomaterials;
2. The HISENTS platform will have widespread applications in addition to nanosafety;
3. It enables screening systems to be fully computer controlled increasing their reliability and reproducibility;
4. The platform is very amenable to PBPK modelling in effect developing the animal-on-a-chip toxicity testing technology backed up by computer prediction;
5. The approach has derived quantitative structure activity relationship ((Q)SAR) properties of NMs so that very general principles of bio-nano-interaction have been derived;
6. HISENTS has created a technology with high translational potential to the pharma, water, health and safety and security industries.

HISENTS overall objectives have been:
1. Synthesised/characterised NM for use as standard test analytes;
2. Designed/tested effective screening devices, each representing a particular physiological function which have been incorporated into a multimodular platform for sensing nanotoxicity;
3. Configured robust electrochemical & optical techniques to interrogate the individual devices.
4. Developed/innovated instrumentation for interfacing to sensor chips;
5. Incorporated/integrated the screening devices into miniaturised and effective flow systems for HTS of NM along a directional pathway;
6. Developed smart automated signal processing data recognition techniques;
7. Carried out comprehensive performance evaluation of platform with respect to each individual device and the whole platform.
Work performed during Periods 1 & 2 (April 2016 to March 2019) is given under individual work packages. WP1 has synthesised 4 groups of NM: Au, SiO2, FexOy and TiO2. These NM were extensively characterised & sent out to all partners in the consortium. In addition Au and Ag NM of varying size, shape and functionality have been synthesised/characterised in particular to understand their interaction with the biomembrane module sensor element. WP2 has developed microfluidic networks and electronic systems for interfacing to the sensor element modules developed by individual partners. In particular a signal processing and data unscrambling algorithm has been successfully developed for analysing data generated by the biomembrane module. The biomembrane module has also been incorporated into an automated fluidic system which together with the electronic interrogation is entirely computer controlled. WP3 has identified strains of miRNA which are sensitive to NM damage. At the same time DNA-lipid conjugates have been incorporated into the biomembrane sensing element so that DNA damage can be electronically detected on-chip. WP4 has carried out a comprehensive study on interactions of novel NM with the biomembrane sensor element in the flow system. The lung-on-chip sensing device was initially configured and the single cell, liver-, kidney-, intestine- and placenta-on-chip devices have been developed together with their associated chip-based supports, flow systems and electronic/optical interrogation. WP5 has developed a 9 compartment PBPK model to mirror the experimental platforms. The model has been tested with published results on NM accumulation in in-vivo and in-vitro tissues and the fit to experimental data is good. WP6 has carried out 2 intercalibration exercises: Exercise (1) three soluble toxicants were sent out to 6 consortium partners. Results were extremely encouraging and showed that all sensor elements responded identically to the toxicity ranking of the toxicants. This can be explained by the fact that the cell/tissues sensor elements were recording membrane damage in their toxicity assay. Exercise (2) four groups of NM were sent out to the same partners. Results were less straightforward due probably to aggregation of the NM with time but they did show some consistency when this was taken into account. WP6 has continued with a general intercalibration of all platforms as well as performance characterisation/testing against the PBPK platform.
HISENTS advances/expected results:
1.Pioneers synthesis of new types of NM. Novel NMs are used to understand more carefully structure-activity relationships and individual pathway analyses of each specific material in multimodular system;
2.Programme included miniaturisation, automation and integration of in-vitro devices into a chip based multimodular smart screening platform;
3.Developed several novel endpoints, including: (a) membrane modification (b) cell deformability change (c) impedance change in cells and (d) DNA structural damage;
4.Took DNA transforming their interrogation to high throughput multimodular devices;
5.Substituted well plates for a flow module interconnected by a microfluidic network that provided each compartment with necessary reagents/media components;
6.Modified the chip-based transducer design miniaturising it from macro to micro/nano dimensions and reconfigured the interrogation system to allow for the highest sensitivity;
7.Integrated a series of high level toxicity assays into a multimodular platform within a microfluidic system where each module contains a sensor element at a successively higher level of biological organisation;
8.Interfaced multimodule flow-through system with smart customised multichannel electronic potentiostatic instrumentation which synchronised flow system manipulation with sensor element handling and allowed several modules to be cross-examined simultaneously;
9.Developed a conceptual framework of the PBPK model approach side by side with experimental systems.

Potential impact:
The global HISENTS technology rationalised current toxicity testing allowing them to be carried out by a few instruments rather than by a whole series of “wet” tests. HISENTS technology revolutionised the way toxicity testing is carried out.
Wider socioeconomic impacts of HISENTS arise from its capacity as a generic automated toxicity screener for appropriate organisations, for instance:
-Water industry: toxins/NM in potable water;
-Environmental/marine agencies of toxins/NMs in the river, estuarine and marine environments;
-Government agencies/statutory bodies: toxicants/NMs in aqueous environments in particular to develop risk assessments for the NMs' use;
-Commercial/academic NM producers for formulation of risk assessment procedures and disposal methods for the materials; and,
-Defence/security industry throughout the EU including NATO, border control and civil defence for the toxicity of NMs where these are manufactured with terrorist ambitions.
Photo of the test setup for simulation of the control module operation with 7 open valves and an act
Prototype TEER microfluidic chip in experimental setup, cell viability A549 cells
Microfluidic module with Si microhole chip, Si microcavity and SEM membrane with microholes
Interactions of increasing aspect ratio Au NM with the membrane module
New biomembrane module. Diagram and photo of flow cells and total platform
HISENTS demonstrator unit shown at IndTech 2018
HISENTS Coordination Team, Exhibition Stand, EuroNanoForum 2017, Valetta, Malta, June 2017
White light (top) and UV light (bottom) optical images of SiO2 _FITC colloidal solns at diff Concs
AFE system in its test run alongside with JTAGICE3 programmer.
TEM images of control and TiO2NPs-, Fe3O4NPs- and AuNPs-treated TH1 cells exposed to equimolar conce
Screenshot of ‘Diagram’ tab/window of microfluidic pump software.
Miniaturised version of comet assay with 12 mini-gels on one slide for measurements of DNA breaks
Schematic showing blood flow-limited PBPK model with 8 compartments.
Cultivation of TH1 cells under a constant flow 20 µl/min of culture medium.