Colored semi-reflective layer for the detection of enzymatic activities – Reflex

The increasing interest in the global climate and in the reduce availability of petroleum resources, added to the human health preoccupations such as a better understanding of the gut metagenome involved in dietary fibber breakdown, have generated a vast amount of research over the decades due to industrial potential in a more efficient utilization (and modifications) of vegetal biomass. These worldwide concerns require the discovering of new enzymes from plant products transformation and the understanding their degradation mechanisms. To achieve this goal, the development of easy-to-use, sensitive and rapid screening tests for high-throughput assays, applicable to a wide range of hydrolytic enzymes is being of crucial importance.
We present an innovative and recently patented screening assay developed in 2010 by INRA1. This assay is based on coloured semi-reflective thin films coated on solid surface and composed of biopolymers. Colour modulation in coating materials is usually achieved by adding dyes or pigments, but can also arise from appropriate structuration of materials. Structural colour can be produced in synthetic materials if the appropriate architecture is built-up in a controlled manner. For instance, this takes place when thin films of controlled thickness in the range of the nano scale and low roughness are deposited on a reflective substrate. Indeed, when incident light reaches an air-film interface, part of it is reflected back while the rest is transmitted into the film while a second reflection occurs at the film-substrate interface. The intensity of the net reflected light depends on the combination of reflected light waves from the air-film and film-substrate interfaces. As a result, the film colour can be tuned by the film thickness. Nanostructured films based on biopolymers can also produce such optical properties. Hydrolysis of the films by the appropriate enzymes induces a decrease in layer thickness and a consequent change in film colour enabling us a visual detection of enzyme activity. Due to the nanometric dimensions of the layers, the method was up to 200 times more sensitive than the classical colorimetric method while the required product amounts and time of analysis were much lower than with the standard methods for screening enzyme activities. The aim of the present project is twice : (i) to propose an in-depth understanding of the enzymatic degradation of the biopolymers within the films as model of the action of enzymes in heterogeneous and dense media, (ii) to develop assays by the fabrication of 96 or 384 wells plates compatible with fully automated strategy in high-throughput approaches.

1. Cerclier, C.; Cathala, B. Méthode de détection d’activités enzymatiques hydrolytiques sans marquage à base de couches réflectives de biopolymères. 2010.