The on-growing importance of personalized medicine and rapid expansion of cancer in vitro diagnostics, generate a strong demand for new biosensors specifically detecting disease biomarkers. Herein, biosensors (or probes) for fluorescence in situ hybridization (FISH) of DNA and RNA biomarkers directly in a single cell, are particularly important to assess the disease and its progression. Current techniques of FISH analysis are long, complex, expensive and comprise complicated amplification protocols to obtain sufficient fluorescence signal. Based on our ultrabright DNA-functionalized dye-loaded fluorescent polymeric nanoparticles (Melnychuk and Klymchenko, 2018; Reisch et al., 2014, 2015, 2017; Trofymchuk et al., 2017) we proposed within the ERC POC project AmpliFISH to develop a simple and fast direct detection of mRNA inside the cells. Thus, the specific objectives were the following: (i) Development of optimized FISH nanoprobes for detection of cancer nucleic acid biomarkers in cells. (ii) Validation of FISH nanoprobes in cancer cell lines with superior performance relatively to standard FISH. (iii) Simultaneous detection of multiple cancer markers within the same single cell. (iv) Validation in clinically relevant samples. (v) Development of a commercialization strategy for the obtained FISH nanoprobes.
Optimization of design and production of FISH nanoprobes was one of the main core activities of this project. First, we found that smaller size NPs (below 20 nm) were essential for free diffusion inside fixed and permeabilized cells, in order to access to the intracellular mRNA targets. Second, proper selection of the polymer matrix of NPs was determined to minimize nonspecific intracellular interactions, which could minimize the off-target nanoprobe binding. We fabricated nanoprobes that can hybridize with different mRNA genes (survivin -BIRC, actin and polyA tails), so we designed corresponding capture sequences and synthesized the DNA-modified nanoparticles for their use in the detection of intracellular RNA. In order to perform multi-color detection of target mRNA, we prepared NPs of three different colors (green, red and far red). We demonstrated that encapsulation of cyanine and rhodamine dyes with bulky counterions yielded green, red and far-red emitting NPs that were 2-100-fold brighter than corresponding quantum dots. These NPs enabled multiplexed detection of three mRNA targets simultaneously within the same single cell and the approach was validated on three cell lines. It revealed unique mRNA expression and special distribution profiles in each tested cancer cell. This achievement is of outmost importance to position our technology in the field of spatial transcriptomics. Additional image analysis confirmed the single-particle nature of the intracellular signal, suggesting single-molecule sensitivity of our method. AmpliFISH was found to be semi-quantitative, correlating with RT-qPCR. Further studies are being performed to improve the quantitative status of our method. Owing to their high brightness, our FISH nanoprobes can detect target mRNA in fixed cells using a simple and rapid protocol (< 3h). Thus, in comparison with the commercial Locked Nucleic Acids (LNA)-based FISH technique, AmpliFISH provides 8-200-fold stronger signal (dependent on the NP color) while requiring only 3 steps compared to the commercial FISH comprised of ~20 steps before microscopy accompanied by multiple washes between each step. However, more research is needed to adapt our methodology to more complex models, such as the clinical samples. On the other hand, we validated the stability and functionality of our DNA-nanoprobes in complex media such as cell lysates. Moreover, we showed that our NPs allows detection of RNA outside the cells in some clinical samples.
The AmpliFISH project has demonstrated unprecedented application of DNA-nanoprobes in the detection and imaging of mRNA cancer markers in single cell level. This work was published in a top peer-reviewed journal in the field of nanotechnology (ACS Nano). Moreover, some findings of this project contributed to preparation of a patent application about a new method for the RNA detection.
The pioneering technology of our nanoprobes allowed the creation of the start-up BrightSens Diagnostics in January 2022, which was in line with original strategy for their commercialized proposed with ERC POC project. The start-up has already won a prestigious French funding for innovative technological companies (i-Lab) and funding from private investor Fondation Force. Moreover, the company obtained a required license on exploitation of all relevant patents of this technology. By creating this start-up, we established a strategy to bring our innovative technology of DNA-nanoprobes to the market of in vitro diagnostics in the coming years. Our nanoprobes will simplify and decrease the cost of RNA detection protocols, currently used in clinical practice, which is the key to improve early diagnostics of diseases and patient follow-up. Thus, the startup will provide a clear position of our technology in the field of human health, in particularly in cancer diagnostics and personalized medicine.
