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

Periodic Reporting for period 4 - NANOZ-ONIC (Bio-inspired electrONIC NOSE interfacing olfactory electrical biosensors and carbon NANOtubes)

Reporting period: 2021-04-01 to 2022-03-31

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.
In the majority of cases, diagnosis of such pathologies occurs in late stages when patients consult their physician due to the appearance of symptoms.
A 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.
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).
With the development of micro- and nano-electronic systems, the concept of electronic noses (e-noses) emerged for the 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.
Unexpectedly, detection of 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 being affected by 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 aimed to develop technologies toward the design of bio-inspired e-noses that mimics the natural olfactory systems. The concept was 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 was performed by international leading experts in these technologies working in the French CNRS Institute and the Korean Seoul National University with the key support from ERC.
The ambitious objective of the 5-year project was 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 were interfaced into the swCNT-FET nano-electronic system for the simultaneous and extremely sensitive detection of volatile compounds. 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.

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 work performed during the project was simultaneously focused on three technical aspects: 1) development of receptor-based biosensors for detecting volatile compounds; 2) optimization of the biological material for interfacing the biosensors with carbon nanotube Field-effect transistors (FETs); and 3) improvements of FETs with higher sensitivity, reusability and a surface appropriate to the analysis of gaseous samples.

The results led to the publication of 26 articles and a book chapter. The project and results were also presented in two joint symposia between the European Federation of Biotechnology and the Asian Federation of Biotechnology.

The main progress obtained to date are:
1) in biosensor engineering:
- The development of a second generation of hybrid receptor/ion channels biosensors with enhanced signal amplitude (Garcia Fernandez et al. Sci Rep. 2021).
- The functional characterization in Xenopus oocytes of optimised human olfactory receptor provided by the laboratory of Professor Matsunami (Duke University, NC, USA) and a fish olfactory receptor.
- The electrophysiological detection of odours with olfactory receptors from other species, widening the diversity of recognized ligands.
- Extracellular domains of taste receptors were successfully used as biosensors demonstrating the feasibility of this approach (Jeong et al. ACS Appl. Mater. Interfaces 2022).

2) in production of biological materials:
- An additional and simpler method than the nanovesicles preparation from mammalian cell membranes has been developed. This method is based on the production of olfactory receptors in bacteria. For their integration in FETs, the receptors were solubilized and stabilized either in nanodiscs (Yang et al. ACS Nano 2017; Lee et al. Sci. Rep. 2018, Lee et al. Biosens. Bioelectron. 2020) or in dual detergent micelles (Shin et al. Sci. Rep. 2020 and Yoo et al. Sens. Actuators B Chem. 2022) for detecting odorants or toxic gas.

3) in the upgrading of FETs
- A magnetic biochip was developed to improve the speed and sensitivity of detection (low non-specific binding) and to facilitate the regeneration of the surface (Yoo et al. Nanotechnology 2018).
- A sol-gel matrix was developed on the FET surface to allow the detection of volatile compounds (Kim et al. Sci. Rep. 2018)
- The addition of engineered floating electrodes enable the specific, quantitative and high sensitivity detection of compounds (Pham Ba et al. ACS Appl. Mater. Interfaces 2018).
- Hybrid surfaces with carbon nanotube and gold reduced noises and resistivity (Shin et al. Adv. Electron. Mater. 2020).

4) in the development of non-invasive detection of viruses
- Development of reusable FET transistors for the detection of the H1N1 virus (Yoo et al. Biosens. Bioelectron. 2020) with increased sensitivity (Shin et al. Sci. Rep. 2021).
- Production of biological materials ("ACE2 antibodies") for the characterization of SARS-CoV-2 virus pseudoparticles. Chevillard et al. Mol. Ther. 2022.
A main progress made beyond the state of the art was the production of mammalian olfactory receptors in a bacterial system, their purification in solubilized and active form, and their successful integration in FET for the detection of compounds. This approach has the potential to greatly facilitate the development of e-noses by simplifying the production of olfactory receptors.
A second main progress is the development of improved FETs with higher sensitivity, a reusable system and a surface enabling the detection of gaseous samples. These improvements are essential for developing FET-based e-noses aiming at detecting low-abundant volatile biomarkers in reusable in vitro diagnostic systems.
Finally, a second generation of biosensors has been developed with much higher amplitudes of the electrical signal. This improved signal amplitude will facilitate the design of biosensors based on human olfactory receptors that are weakly expressed. Preliminary results with non human receptors are encouraging for developing new biosensors.