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Bio-inspired electrONIC NOSE interfacing olfactory electrical biosensors and carbon NANOtubes

Periodic Reporting for period 3 - NANOZ-ONIC (Bio-inspired electrONIC NOSE interfacing olfactory electrical biosensors and carbon NANOtubes)

Reporting period: 2019-10-01 to 2021-03-31

Early detection of diseases is a key factor for their rapid and efficient treatment, and this is even more crucial for pathologies with high rate of mortality such as cancers and neurodegenerative diseases. Thus, epidemiological studies demonstrated that the 5-year survival rate of patients with lung cancers was 58-73% when detected in stage I, which drops to 3.5% in later stages (Taivans et al., 2014).
In the majority of cases, diagnosis of such pathologies occurs in late stages when patients consult their physician due to the appearance of symptoms. Then efficient methods exist to diagnose such pathologies by blood tests, biopsies, chest X-ray radiography, computed tomography, positron emission tomography or magnetic resonance imaging.
However these methods are either invasive or irradiative or massive and require technical expertise. Consequently they are not suitable for routine examinations on a large cohort of patients which is fundamental to widely diagnose diseases in early and asymptomatic stages.
The central societal and medical challenge in the field is to develop technologies that would safely and non-invasively detect pathologies such as cancers as easily as the blood pressure test that is systematically performed during medical consultations.
A fascinating and highly promising approach is naturally inspired by the discovery of dogs detecting some human cancers with their sense of smell. This discovery revealed that specific odors are generated by tumor cells and can be detected by an olfactory system. Some worldwide Medical Centers (e.g. In Situ Foundation; Medical Detection Dogs) and companies (e.g. KDOG) exploit now the ability of trained dogs to diagnose specific cancers in early stages by sniffing samples.
However, the use of dogs has some limitations mostly due to the long period of training (50 dogs were trained in 12 years by the In Situ Foundation), the incapacity to deploy them on every medical offices, their high costs (£ 29 000 / dog: www.medicaldetectiondogs.org.uk) and some cancers like prostate cancer cannot be accurately detected by dogs in samples like urine (Dorman et al., 2017).
However they reliably detect lung cancer from patients' breath (Fischer-Tenhagen et al., 2018), and breath is an attractive sample since it is non-invasive, simple to collect repetitively and more importantly at least 17 pathologies were identified as producing detectable volatile biomarkers (Konvalina and Haick, 2014). The pathologies compatible with in vitro diagnostics of breath analysis include airway-related diseases (asthma, lung cancer, chronic obstruction of pulmonary diseases, cystic fribrosis), but also cancers from various organs (e.g. breast, colorectal, head and neck, prostate cancers), metabolic diseases (e.g. diabetes mellitus, hepatic and chronic kidney diseases), neurodegenerative disorders (e.g. Alzheimer's and Parkinson's diseases) and bacterial infections (e.g. tuberculosis, pneumonia, Helicobacter pylori).
With the development of micro- and nano-electronic systems, the concept of electronic noses (e-noses) emerged forthe detection of volatile organic compounds (VOCs) through electrical, gravimetric or optical signals in handheld devices. Different technologies of e-noses were developed and the most advanced technologies are based on "chemiresistors" that recognized VOCs by physico-chemical interactions. A commercialized device already exists (www.aerocrine.com) for diagnosing and monitoring asthma via the accurate and highly sensitive detection of nitric oxide (NO), a volatile biomarker presents in the patient's breath.
Unexpectedly, the extension of this technology to other diseases such as cancers appeared to be more complex because for most diseases, not a single biomarker is sufficient to obtain an accurate diagnosis. Therefore, the technological challenge is to simultaneously and quantitatively detect several biomarkers with high sensitivity, high specificity and without sensitivity to confounding factors like humidity, temperature and pressure. While the physico-chemical biosensors are highly sensitive and suitable for gas analysis, they are also poorly specific, weakly versatile and highly sensitive to confounding factors.
In order to develop a new generation of e-noses that overcomes these technological obstacles and complement the current range of biosensors, the NANOZ-ONIC project aims to create bio-inspired e-noses that mimics the natural olfactory systems. The concept is based on two cutting-edge technologies: 1) artificial receptor-ion channel proteins that generate an electrical signal when a receptor recognizes a specific compound; and 2) the highly sensitive and miniaturized single-wall carbon nanotube field-effect transistors (swCNT-FET) that are interfaced with the artificial receptor-ion channel proteins. The project is executed by international leading experts in these technologies working in the French CNRS Institute and the Korean Seoul National University with the critical support from ERC.
The ambitious objective of the 5-year project is to design a library of olfactory biosensors based on olfactory receptors which would provide the required high specificity and versatility for diagnosing new pathologies by breath analysis. The biosensors will be interfaced in an array of different receptors into the swCNT-FET nano-electronic system for the simultaneous and extremely sensitive detection of biomarkers.
The long term objective of the project is to create a hand-held device able to detect volatile biomarkers from exhaled breath samples and alert to the presence of diseases in early asymptomatic stages. The portable devices would be used for point-of-care analysis either in medical institutions or even at home thanks to the simplicity of the sample analysis, the real-time readout and their integration in electronic devices able to transmit data through existing communication networks. Moreover the undemanding sample collection would also facilitate the monitoring of treatment efficacy and help to rapidly adjust the posology.

References:
Taivans et al. (2014) Expert Rev Anticanc 14, 121-123.
Dorman et al. (2017) Vet Med. 8, 69-76.
Fischer-Tenhagen et al. (2018) Front Vet Sci 5, 52.
Konvalina and Haick (2014) Acc Chem Res 47, 66-76.
The design of the olfactory biosensor library was initiated rapidly after the recruitment of the French team dedicated to the project in Oct. 2016. Five olfactory receptors have been already fused to the ion channel in order to create the first biosensors. These receptors recognize heptanal, a volatile biomarker of lung cancer, and trimethylamine, a volatile biomarker of the metabolic disease trimethylaminuria. Artificially attaching two proteins together is not sufficient to create functional biosensors. Precise protein engineering is required to induce a functional "communication" between the receptor that recognize the molecules and the ion channel that generates the electrical signal. Several engineered biosensors were created for each olfactory receptor and they were heterologously expressed in the batracian Xenopus oocytes for their electrophysiological characterization. However the notorious low expression of olfactory receptors in other cells than the natural olfactory sensory neurons impedes their characterization in Xenopus oocytes. Intensive efforts were made to boost the expression of the olfactory biosensors and the first encouraging results was recently obtained with one olfactory receptor. More efforts are ongoing especially to transform the Xenopus oocytes in efficient cells for expressing the olfactory biosensors.
In parallel, we are developing innovative strategies of biosensor design with 4 objectives: 1) to avoid the time-consuming step of protein engineering; 2) to develop high-through screenings for testing the expression and the function of olfactory biosensors; 3) to simplify the production of nanoparticles containing the biosensors; and 4) to improve the properties of the biosensor library with other ion channels.
Simultaneously, improvements and innovations are made on the nano-electronic platform in order to develop reusable sensors, to increase the transducer sensitivity and to create gas permeable surfaces. The positive results obtained with biological sample models will be exploited for interfacing the olfactory biosensors.
Instead of conducting a single strategy for the design of the biosensor library, we developed several different approaches thanks to the important human workforce dedicated to this project. These innovative approaches have a great potential of biosensor production. Consequently, we expect at the end of the project to go beyond the initial objective in terms of number of biosensors integrated in the nano-electronic platform. Every biosensor added in the nano-electronics platform will increase the diagnosis accuracy and the panel of detectable diseases.
The French and the South Korean laboratories are joining their efforts to succeed in the design of an e-nose integrating the olfactory biosensor library.
In case of success, a bigger consortium of diverse expertise will be created in a subsequent project in order to develop this technology toward medical applications for in vitro diagnostics using breath analysis.