Enzymatic Commutators for Specific and Ultra-Sensitive Sensing – ECOSENS

The ultimate challenge of molecular sensing is the detection of ultralow concentrations of a specific analyte in a complex mixture using fast, simple and easy to handle analytical tools. The main objective of the present project is to develop a new generic analytical technology able to reach ultra-low detection limits of organic, inorganic or biological analytes in environmental or biological samples, with an improved simplicity and cost-efficiency.
Our strategy relies on the unprecedented combination of molecular and enzymatic switches coupled with electrochemical methods for highly sensitive detection of small analytes and will be validated with targets as H2O2, fluoride, or H2S. Such detection is of great importance because those molecules plays important roles in various disease or biological functions: excess amount of fluoride causes fluorosis, urolithiasis, or even cancer; elevated levels of hydrogen peroxide have been found in a number of respiratory disorders as well as in renal dysfunction; unregulated, abnormal levels of H2S may contribute to various diseases since its implication is now well established in the cardiovascular domain, in the control of inflammation, in the central nervous system as a neuromodulator or neuroprotectant, and in cellular bioenergetics, with relevance to hypoxia sensing or tumor proliferation.
The key idea is to design molecular switches which combine a target-triggered autocatalytic reaction and the release of a cofactor for the activation of a surface confined apoenzyme. This strategy would provide unprecedented sensitivity since it adds up two amplifications. The first is the autocatalytic amplification of the small analytes coupled with the exponential release of the cofactor from a suitably designed molecular pro-cofactor. The second amplification is based on the electrochemically-monitored activation of a surface confined inactive apo-enzyme thanks to the release of the cofactor in the previous reaction. The biocatalyst activation allowed so far detection of picomolar concentrations of the cofactor in solution and femtomolar concentrations of analytes should be detectable from the combination of the two amplifications.
This strategy will be then advantageously adapted to design ultrasensitive immunoassays with the upstream addition of a fluoride or H2O2 producing enzyme. These small molecules will be considered as intermediate products generated by an enzyme label in an affinity binding immunoassay of ELISA type. An ideal gain of sensitivity close to 1 million over classical ELISA is projected. Developing more sensitive technologies will provide a great step forward in clinical diagnosis. For instance, lowering the detection limit of biomarkers would provide a great tool for several applications as the early detection of cancer and cancer recurrence, the early detection of infectious disease or neurologic disorder, or the identification of new biomarkers. Our project thus aims at reaching similar low detection limits of protein biomarkers as those achieved with the scarce number of commercialized ultrahighly sensitive immunoassays, but with a much lower cost, simplicity, and a greater adaptability to existing technology and laboratory practices.
Important efforts will be made to design and synthesize the pro-cofactor in a highly pure form. Evaluation of their ability to release the cofactor together with autocatalytic amplification of the initial low amount of the target will be crucial for the later development of this innovative technology.
Presented in the challenge “Renouveau du tissu industriel” (Challenge 3, White biotechnology and bio-inspired chemistry; orientation 15: Sensors and Instrumentation), the overall objective of ECOSENS is the deployment in the market of modular sensing platform for the accurate detection of multiple compounds of interest (from small molecules to proteins) in various samples, with time to market realistically within five to ten years.