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Integration of living cells with organic transistors for the rapid detection of toxins and enteric pathogens

Final Report Summary - CELLTOX (Integration of living cells with organic transistors for the rapid detection of toxins and enteric pathogens)

The epithelium plays a significant role in resistance to infection in mammals, and is made up of a single layer of elongated, column-shaped cells that line the stomach and colon. This single layer of epithelial cells restricts the entry of toxins and pathogens, while selectively absorbing nutrients that sustain the body. Pathogens have devised multiple mechanisms to destroy the integrity of the intestinal epithelial barrier, compromising the normal absorption of water in the intestine and thereby causing diarrheal disease. The World Health Organization estimates that in 2005 alone 1.8 million people died from diarrheal diseases. CELLTOX is a novel type of biosensor for detection of enteric pathogens and toxins, based on the principle of using live epithelial cells grown on an organic electrochemical transistor (OECT), which provides a very sensitive and convenient means of measuring ionic transport. When the epithelial cells form a monolayer, the integrity of the cell monolayer blocks ion transport keeping the transistor in the ON state. Assault of the cells by an enteric pathogen or toxin will lead to a disruption of the cell monolayer and enable ion migration into the polymer, switching the transistor OFF. This novel “canary in a coal mine” platform will constitute a broad first-line diagnostic for gastrointestinal disease, with applications for food and water safety. It will lead to sensors that are fast, portable, inexpensive and label-free. Future use of different cell lines (e.g. bronchial, dermal, etc.) with this platform will lead to a host of sensors for applications in medical diagnostics, agriculture, and environmental protection. This multidisciplinary project encompasses the disciplines of organic electronics, cell biology and microbiology, and will contribute to the successful and lasting reintegration of the applicant back to Europe.

The goals as outlined in the Celltox project were to:
1. Asses the formation of epithelial cell monolayers on conducting polymers
2. To design and fabricate Organic electrochemical transistors (OECTs) for sensitive detection of ionic currents
3. To integrate epithelial cell monolayers with OECTs
4. To evaluate the performance of the OECT-epithelial cell devices for detection of pathogens and toxins.

The ability of organic electronic materials to conduct both electronic and ionic carriers makes them ideally suited for interfacing with barrier tissue cells. During this project, we demonstrated the integration of human epithelial cell barrier tissue layers (Caco-2) with OECTs as a means of assessing barrier tissue integrity. We explored the best way of integrating cells with the conducting polymers and discovered an easy method that is in line with current cell culture techniques in laboratories around the world. We demonstrated that we see a clear difference between devices with cells integrated, and a device with no cells integrated. We showed that the OECT can measure disruption in barrier tissue caused by a variety of toxic compounds, with inherent amplification from the transistor resulting in sensitivity greater or equal to the other methods tested. We then went on to validate our technology by comparing it with conventional methods used to assess the integrity of epithelial cell layers: immuno-fluorescence, permeability assays, and electrical impedance scanning. We demonstrated that a variety of toxins and pathogens, including the food borne pathogen Salmonella typhimurium, induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. To demonstrate feasibility for diagnostics applications, the sensors were scaled up to a 4-plex format, with possibility for simultaneous operation, under physiological conditions in a cell-culture incubator. Stable operation of the organic devices under physiological conditions was enabled with dynamic, pathogen-specific diagnosis of infection of epithelia by the bacteria. Operation of the device was also demonstrated in milk, demonstrating feasibility for diagnostics in complex matrices, showing particular promise for food and safety applications. Due to the potential of low cost processing techniques and the flexibility in design associated with organic electronics, the OECT has great potential for high-throughput, disposable sensing and diagnostics.
- a description of the results achieved in the CellTox project:
• We have successfully integrated epithelial cell layers with conducting polymer devices
• We have developed an OECT sensor that is capable of measuring minute changes in ion flow
• We have successfully monitored disruption of epithelial cell layers integrated with OECT
• We have shown highly sensitive and toxin/pathogen specific diagnostics using the epithelial cell layers integrated with the OECT

We have demonstrated that the OECT compares very favorably with other methods tested as a highly sensitive, dynamic system for measuring epithelial cell layer integrity, with increased temporal resolution and sensitivity than conventional methods, in the case of all the toxins and pathogens tested. Importantly for diagnostics, the OECT is stable under physiological conditions, can operate for long periods and further, operates competently in complex matrices such as milk, which often cause problems for conventional diagnostic methods. The work carried out in the Celltox project has fulfilled all of the aims of the project and will be continued with other leveraged funding in an effort to produce a working prototype for commercialization purposes.
- the expected final results and their potential impact and use
The OECT-epithelial cell sensor has been demonstrated to be a highly sensitive, dynamic sensor for in vitro diagnostics of pathogens and toxins. The nature of the device means that the design may easily be changed and modified to suit a particular application, especially if called for by constraints in cell culture, such as for co-culture models. Further optimization of the OECT is ongoing to increase sensitivity to provide even greater insight into the disruption mechanisms of toxins and pathogens on barrier tissue. OECTs have also been demonstrated to be to a very accurate and reliable monitoring system for the development of in vitro cell models for toxicology purposes, thus generating a system which is inexpensive, rapid and reduces animal experimentation. This is prompted by the urgent need to move away from using animal models for toxicological testing as well as pharmacological evaluation of chemical substances, driven both by societal distaste and also the large cost of using animals. The validity and predictive ability of in vitro based models has been shown to be poor and results in significant failures of drugs with a high associated price-tag. One of the major benefits from a switch to the use of in vitro models with associated accurate monitoring systems will be the ability to perform high-throughput screening with low associated costs, which is not currently the case. The OECT shows excellent potential for low cost fabrication, and thus high-throughput processing of samples. A prototype is currently under development in our laboratory that will demonstrate this principle (funded by the National Centre for the reduction of experimentation on animals; NC3Rs, UK). Work has also begun on the use of different cell lines (e.g. bronchial, kidney, etc.) with this platform will lead to a host of sensors for applications in medical diagnostics, agriculture, and environmental protection.