Funding type: COFUND Marie Slodowska Curie Action
This project is under the Marie Skłodowska-Curie Actions (MSCA) program. There are no nationality conditions but the candidates must fulfill the MSCA mobility conditions, which means that she/he must not have stayed more than 1 year in France during the last 3 years immediately before the call deadline (31/05/2019)

Expected start date: 01/10/2019

Contacts:

Ass. Prof. Emmanuelle Laurenceau, This email address is being protected from spambots. You need JavaScript enabled to view it.
+33 4 72 18 62 40

Dr. Cesar Sanchez Huertas, This email address is being protected from spambots. You need JavaScript enabled to view it.

websites:

• https://www.rmit.edu.au/about/our-locations-and-facilities/facilities/research-facilities/micronano-research-facility
• http://inl.cnrs.fr/en/

Collaborations/External partners: Montpellier Hospital

Scientific context: Breast cancer is the second most common cancer diagnosed worldwide, so it still remains a major public health problem. 90% of breast cancer-related deaths are caused by distant metastases. Thus, disseminated malignancy remains one of the main diagnostic and therapeutic challenges today. However, early dissemination of tumour cells is usually undetectable in patients by conventional histopathology, molecular and imaging techniques. Numerous cancer biomarkers have been identified. Among them, serum circulating cancer biomarkers (CCB) (tumor proteins and circulating miRNA) are promising biomarkers for cancer prognosis and treatment monitoring. Currently, the detection of these biomarkers (qPCR, ELISA, MS, etc.) remains time and sample-consuming, expensive and complex, which difficult their analysis in a clinical routine basis. In this regard, biosensors stand out as promising technology solutions for CCB detection due to their selectivity, sensitivity, and their capabilities for miniaturization for the development of simple-to-use Lab-on-chip and Point-of-care devices. Biosensors can be designed to provide quantitative analytical information with elevated accuracy in a few minutes, using low sample volumes and minimum sample pre-treatment. Recent breakthroughs in integrated circuit technology have led to the creation of new types of biosensors based on photonics (Fernández Gavela, Grajales García, Ramirez, & Lechuga, 2016) showing very low limits of detection (Huertas, Fariña, & Lechuga, 2016; Huertas & Lechuga, 2017). These new biosensors can be fast, cheap, easy to use and very accurate and hence have the potential to revolutionise disease diagnosis and treatment allowing conclusive clinical analysis directly at the Point-of-Care (PoC) (Syedmoradi et al., 2017). The goal is to use the devices outside the laboratory for the continuous monitoring of the patient before and after treatment. Such devices may allow the simultaneous detection of multiple biomarkers belonging to a previously defined cancer profile. This early diagnosis will define a newer and hopeful starting point for cancer treatment, helping design specific protocols and targets for therapies specifically tailored for each patient.

Key-words: multiplex biosensor; breast cancer; cancer circulating biomarkers; diagnosis; prognosis; lab-on-chip; personalized medicine.

Objectives of PhD thesis:

The subject of the PhD thesis aims at developing cost-effective and easy-to-use lab-on-a-chip platforms as point-of-care devices to be used in routine screening and contribute to the development of personalized cancer diagnosis and treatment. This device will be based on an array of photonic biosensors able to detect circulating cancer biomarkers of diverse nature, such as tumor proteins and miRNA in order to achieve a more complete and reliable diagnosis, prognosis and monitoring of metastatic breast cancer.

Scientific hurdles:

CCB biosensors generally focus on only one type of biomarker and their clinical validation is not efficient. In this project, we plan to elaborate an integrated biosensor combining different recognition platforms:

• DNA sensor for miRNA detection.
• Antibody sensor for tumor antigen detection.

For each recognition platform, the set of biomarkers should be identified in order to improve the specificity of the detection. The integration of these various recognition platforms needs the combination and integration of different surface chemistries to achieve selective and specific biofunctionalization on the same microsystem. This point is particularly crucial for the efficient multiplex detection of circulating cancer biomarkers.

Moreover, the photonic detection device will consist of a Photonic chip that can contain different biosensors for multiplexed analyses. Each biosensor could have immobilized different biorecognition elements that would interact specifically with the biomarkers of interest and give quantitative information in real time of such molecules in a given sample, such as blood. The working principle of this type of biosensors rely on light for the generation of an Evanescent field very sensitive to refractive index changes that take place on the sensor surface. This phenomenon allows for the label-free detection of minute concentrations of these biomarkers of interest just by their interaction with the specific biorecognition elements at the sensor surface.

The chips would be integrated along with a sophisticated microfluidics delivery system that allows the targeted biomarkers to be in contact with the biorecognition element at the sensor surface. We imagine an automated microfluidics system including all the necessary laboratory processes on the same chip, facilitating the manipulation of the sample in a faster way and requiring very low sample volume. This will allow for a more efficient and simplified analysis with faster turn-around times. Finally, the biosensor device will include all the electronics and signal processing system that will translate the biomolecular interactions into easy to interpret signals.

Finally, validation of the integrated biosensor with breast cancer sera from Biobank and qualified with conventional techniques (ELISA, PCR) is also a critical point to implement for the evaluation of biosensor performances (sensitivity, selectivity, reliability).

Innovative contributions:

One major innovation concerns the integration of all the microfluidics and photonics technology in the same platform to guarantee the detection of different biomarkers, i.e miRNA and proteins. To our knowledge, this kind of integrated-multiplex biosensor was never described in the literature and could greatly help clinicians in diagnostic and therapeutic decisions for multifactorial diseases such as cancer.

Another innovation concerns the development of a methodology allowing for multiple surface functionalizations (chemical and biological) on the same surface.

The simplicity of the analysis would allow for the use of this type of devices in clinical settings outside the hospital and closer to the patient for population screening and therapy follow-ups. Due to the integration of all the procedures necessaries for the analysis of such biomarkers, it would make possible to use this technology by non-specialized personnel, saving resources and offering a faster analysis.

Working plan:

The first task will be the design and optimization of recognition platforms. Each recognition platform will be implemented separately to define the optimal conditions (surface chemistry, probe concentration, spotting buffer) for efficient biological recognition:

• miRNA biosensor platform: At first, two of the most relevant miRNA described in the literature for the sensitive and specific detection of metastatic breast cancer will be implemented: miR-21 and miR-155.
• Tumor protein biosensor platform: Based on previous studies, antibodies against circulating tumor proteins will be immobilized on the sensor chip (Yang et al., 2013).

For prognosis and therapeutic issues in breast cancer, tumor proteins targeted will be HER2, uPA and PAI-1 (Shi et al, 2018; Nguyen et al., 2018)
This project will adapt and validate an existing, prototype photonic chip developed in the Integrated Photonics and Applications Centre, led by Dist. Prof. Arnan Mitchell. The prototype photonic chip is fabricated with conventional microelectronic technology and consists of 30 identical Mach-Zehnder interferometers (MZI) fabricated in silicon substrate. MZI biosensors have an input single-mode waveguide that originates two arms after a Y-junction: one of the two arms is exposed to the external medium to be sensed, while the other is employed as a reference. These two arms are recombined through a second Y-junction into a single output waveguide, resulting in an interference pattern with a determined number of fringes of certain amplitude (visibility factor) depending on their sensitivity. Performances of each biosensor platform will be compared to conventional methods (PCR, ELISA).

The second task will concern the integration of the biosensing platforms in one functional microfluidic device. Several micro-structured designs will be elaborated and tested to guarantee the multiplex detection of circulating cancer biomarkers in the same platform. The microfluidic system will be adapted from a first prototype designed by the the InPAC centre (Szydzik et al., 2017), integrating additional microfluidics components necessary for the final Breast-cancer PoC device. The proof-of-concept will be validated with a device integrating 2 to 5 different probes, as described above, and could be implemented with more mi-RNA probes depending on the results to achieve a more accurate diagnosis.

The third task will be the biological validation and clinical evaluation using statistical analysis with breast cancer patient sera obtained from the Hospital biobank.

PhD funding : Co-Fund ECL/RMIT (ECLAUsion program)

Profile of the PhD student:

The applicant should have MSc in chemistry/biochemistry or materials and a strong interest for multidisciplinary research related to human health. He (she) will like experimental work and be able to work at the interface of chemistry and biology. He (she) will not have studied in France for more than 2 years over the last 3 years.

Benefits of the project:

Publications in peer-review international journals and international conferences.

Patent proposal depending on the results.

Skills developed during the PhD:

At the end of the thesis, the PhD student will have acquired skills in chemical and biological functionalization, microfluidic system and photonic technology. He (she) will be autonomous on several characterization methods (IR, XPS, etc). He (she) will participate to the writing of publications and eventually patents, and will present his (her) research work to international conferences.

References:

1. Fernández Gavela, A., Grajales García, D., Ramirez, J., & Lechuga, L. (2016). Last Advances in Silicon-Based Optical Biosensors. Sensors, 16(3), 285. https://doi.org/10.3390/s16030285
2. Huertas, C. S., Fariña, D., & Lechuga, L. M. (2016). Direct and Label-Free Quantification of Micro-RNA-181a at Attomolar Level in Complex Media Using a Nanophotonic Biosensor. ACS Sensors, acssensors.6b00162. https://doi.org/10.1021/acssensors.6b00162
3. Huertas, C. S., & Lechuga, L. M. (2017). Simple, low-cost, and timely optical biosensors for the detection of epigenetic biomarkers: The future of cancer diagnosis. European Medical Journal. Retrieved from https://emj.europeanmedical-group.com/wp-content/uploads/sites/2/2018/01/Editor’s-Pick-Simple-Low-Cost-and-Timely-Optical-Biosensors-for-the-Detection-of-Epigenetic-Biomarkers-The-Future-of-Cancer-Di.pdf
4. Syedmoradi, L., Daneshpour, M., Alvandipour, M., Gomez, F. A., Hajghassem, H., & Omidfar, K. (2017). Point of care testing: The impact of nanotechnology. Biosensors and Bioelectronics, 87, 373–387. https://doi.org/10.1016/J.BIOS.2016.08.084
5. Szydzik, C., Gavela, A. F., Herranz, S., Roccisano, J., Knoerzer, M., Thurgood, P., … Lechuga, L. M. (2017). An automated optofluidic biosensor platform combining interferometric sensors and injection moulded microfluidics. Lab on a Chip, 17(16), 2793–2804. https://doi.org/10.1039/C7LC00524E
6. Nguyen, H. T., Dupont, L. N., Jean, A. M., Géhin, T., Chevolot, Y., Laurenceau, E. & Gijs, M. A. M. (2018). Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides. J. Micromech. Microeng. https://doi.org/10.1088/1361-6439/aaa7a1
7. Shi, L., Gehin, T., Chevolot, Y., Jacot, W., Lamy, P.J. & Laurenceau, E. (2018). Quantification of uPA in breast tumour tissue extracts by microarray immunoassay: Comparison with ELISA technology (2018). Journal of Applied Biomedicine, 16, 214–220. https://doi.org/10.1016/j.jab.2018.01.001
8. Yang, Z., Chevolot,Y., Géhin,T., Dugas,V., Xanthopoulos, N., Laporte,V., Delair, T., Ataman-Önal, Y., Choquet-Kastylevsky,G., Souteyrand,E. & Laurenceau, E. (2013). Characterization of three amino-functionalized surfaces and evaluation of antibody immobilization for the multiplex detection of tumor markers involved in colorectal cancer. Langmuir, 29, 1498−1509. dx.doi.org/10.1021/la3041055