New rapid diagnostic tool for sepsis: microfluidic biochip for multi-target detection by isothermal amplification
Sepsis is among the main cause of death across the world, and is caused by severe bacterial infection but can also originate from viruses, fungi or even parasites. In order to drastically increase survival rates, a rapid diagnostic and appropriate treatment is of paramount importance. The commercially available tools for nucleic acid detection by qPCR are able to sense multiple targets. However, these multiplexed analyses arise from the accumulation of analysis channels or reaction chambers where only one target can be detected. The original sample has to be divided, resulting in a loss of sensibility since a smaller amount of targets is available in channels or chambers.
In order to tackle the question of “How to detect multiple targets without a loss in sensibility?”, the PhD candidate will have to develop a multiplexed detection in a single reaction chamber by localized immobilization of LAMP primers (Loop-mediated isothermal amplification) on a solid substrate like COC or glass.
The expected outcome is a biochip allowing for real-time and fast (minutes) detection of several molecular DNA targets including: primers design and selection, primers immobilization on surface, integration of the biochip into a microfluidic cartridge and data collection and management for fluorescence detection of targets.
This innovative work will provide the PhD candidate with strong skills in diverse scientific domains such as molecular biology, surface functionalization, modelling and simulation, in a very multidisciplinary working environment.
On-line monitoring of bioproduction processes using 3D holographic imaging
The culture of adherent cells on microcarriers (MCs) is a promising approach for various bioproduction applications, such as drug manufacturing and delivery, regenerative medicine, and tracking of cellular differentiation. However, the analysis of single cell morphology and behavior without affecting the substrate integrity remains a major challenge. Lens-free holographic imaging is emerging as a promising solution for real-time, non-invasive monitoring of cellular processes. This technique captures wide field of view images without requiring exogenous labeling or sample manipulation, thus preserving the integrity of the cellular environment.
This thesis proposes the development of a 3D lens-free imaging system to monitor cells on MCs in near real-time. The microscope will be coupled with advanced algorithms for data reconstruction and analysis in on-line bioreactors. The use of deep learning techniques will allow for real-time segmentation and analysis of single cells, facilitating the tracking of cellular dynamics. This innovative project paves the way to a non-invasive monitoring of 3D multicellular samples, with potential applications on organ-on-chip and more complex organoids systems.
Sperm 3D - Male infertility diagnostic tool using holography for imaging and 3D tracking
Infertility is a growing problem in all developed countries. The standard methods for the diagnostic of male infertility examine the concentration, motility and morphological anomalies of individual sperm cells. However, one in five male infertility cases remain unexplained with the standard diagnostic tools.
In this thesis, we will explore the possibility to determine the male infertility causes from the detailed analysis of 3D trajectories and morphology of sperms swimming freely in the environment mimicking the conditions in the female reproductive tract. For this challenging task, we will develop a dedicated microscope based on holography for fast imaging and tracking of individual sperm cells. Along with classical numerical methods, we will use up-to date artificial intelligence algorithms for improving the imaging quality as well as for analysis of multi-dimensional data.
Throughout the project we will closely collaborate with medical research institute (CHU/IAB) specialized in Assisted Reproductive Technologies (ART). We will be examining real patient samples in order to develop a new tool for male infertility diagnosis.
Ultra-wide-field smart microscope for the detection of egg parasite (SCREENER)
In most parasitic cycles, the free phase passes through an egg stage, which is released by the host into the environment via a complex faecal matrix, which has highly variable and often low egg concentrations. The classical detection method relies on microscopic observation of these eggs, which implies a tedious and time-consuming preparation of the sample to concentrate the eggs, with highly variable sensitivity values. This detection is crucial because, once dispersed, the eggs contaminate the environment and food, leading to cases of parasitic zoonoses in humans.
Detection in environmental and food matrices is even more complex than for faeces because of the very low number of eggs present : 1 to 10 per sample in the vast majority of cases. The thesis aims at developing a lensless wide-field imaging system that will allow the counting and identification of parasite eggs in complex matrices, while increasing sensitivity. This will make it possible to automate detection, thus opening up the possibility of investigating more samples for better health surveillance.
Early diagnosis of sepsis using a GMR sensor-based biochip
Sepsis, an extreme and deregulated immune response to an infection that then spreads through the bloodstream, can lead to organ dysfunction and death (11 million deaths worldwide every year). The patented GMR (Giant MagnetoResistance) sensor-based biochip we have developed has real potential for the early detection of pathogens involved in sepsis or biomarkers of the disease, present in very small quantities in the blood, without the need for a culture step. The innovative approach we are proposing is cross-disciplinary, since it is based on the use of magnetic nanoparticles (NPM), functionalized by monoclonal antibodies produced in the LERI laboratory, directed against target biological objects (cells, bacteria, yeasts, etc.) which are detected dynamically and simultaneously one by one by GMR sensors arranged on either side of a microfluidic channel in which they flow. Proof of concept for this biochip was obtained on a murine myeloma cell model.We were able to achieve a sensitivity and specificity with this model that makes our technique highly competitive with existing Point-of-Care tests. However, we still need to validate these results on pathogens.
During the course of the thesis, two objectives will be defined. Following on from the current thesis, the first objective of the student at the LNO will be to adapt the biochip (sensors, microfluidics and signal processing) so that it is sensitive and rapid for the detection of bacteria and yeasts involved in sepsis in blood samples. At LERI, he will optimize the magnetic labeling of bacteria and yeasts in this clinical matrix using commercial NPM functionalized with one or more antibodies directed against the target. This stage of the thesis will be carried out in close collaboration with the Service de Bactériologie et Hygiène at Hôpital Béclère (also a member of the IHU), which will recommend and supply relevant bacterial and yeast strains for detection, as well as clinical samples.One of the GMR biochips will be installed at Hôpital Béclère for measurements under real-life conditions. The second objective will be to use the GMR biochip to quantify the reduction in monocyte expression of mHLA-DR molecules, which is an indicator of the immunosuppressed state of sepsis associated with increased infectious risk and mortality.
Microwave Near Field Sensing in Heterogeneous Media
This thesis focuses on the development of microwave near-field sensing techniques for applications in biomedicine, agronomy, and geophysics. The primary objective is to design low-complexity algorithms that effectively solve complex inverse problems related to the characterization and detection of dielectric properties with various geometric distributions in heterogeneous media.
The candidate will begin by conducting a comprehensive review of existing radar-based and advanced signal processing methods. A precise physical model of microwave propagation in near-field conditions will be developed, serving as the foundation for new detection methods based on the concept of physics-driven iterative tomography. The ultimate goal is to formulate efficient algorithms suitable for real-time applications and validate them through experimental implementation. To achieve this, an evolving prototype setup will be developed, progressing from 2D media to more complex 3D scenarios.
This interdisciplinary project combines physical modeling, algorithm development, and practical experimentation. It presents an opportunity to advance the field of microwave imaging, with significant implications for biomedical and environmental applications.
Development of a microfluidic bioanalytical platform to quantify the cellular bio-distribution of a drug
A drug's mode of action and efficacy are correlated not only with its ability to accumulate in the targeted pathological tissues, i.e. its tissue bio-distribution, but also with its ability to specifically reach its molecular target within cells. Non-specific accumulation of a drug in these cells can be the cause of undesired effects, such as side effects during chemotherapy. In other words, assessing a drug's efficacy, specificity and absence of toxicity requires precise, quantitative determination of its cellular bio-distribution. Antibody-drug conjugates (ADCs) have become an indispensable tool in oncology, enabling vectorized therapy to preferentially target a subset of tumor cells expressing the antigen recognized by the antibody.
These ADCs target specific tumor cells expressing a particular antigen, thus limiting toxicity to healthy tissue. Radioactive labeling of drugs (3H, 14C) is a key method for quantifying their accumulation in tumor and non-tumor cells, in order to assess targeting accuracy and avoid undesirable side effects. However, the detection of low-level tritium emissions requires new technological solutions. The project proposes the development of an innovative microfluidic platform to detect and quantify these isotopes in single cells. This approach will enable us to better document ADC distribution in heterogeneous tissues and refine therapeutic strategies.