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Detecting enzymatic activity: a test in full colour…

A patented device for the detection of enzymatic activity has been developed.  It is based on the colours specific to the nanometric scale in biopolymer films.  Rapid, simple and sensitive, this test could be miniaturised to enable its inclusion in high-throughout screening systems for applications in metagenomics, white biotechnologies, etc.

détection d'activité enzymatique. © inra
Updated on 04/22/2013
Published on 04/12/2013

The principle of the test: the use of colour without staining
Although most of the colours we observe in nature are linked to the presence of coloured pigments in different objects, others only exist through a particular organisation of matter and its interaction with light.  These are referred to as "structural colours".  This is notably the case of the feathers of certain parrots, or the wings of butterflies or beetles, etc.  
Scientists in the Research Unit on Biopolymers – Interactions and Assemblies (BIA) have exploited these structural colours in biopolymer films at a nanometric scale to develop a patented detector for enzymatic activity. They applied biopolymer films about 100 nanometres thick on a reflective substrate (silicon), knowing that at thicknesses between 70 and 200 nm a colour would appear due to interference phenomena. Some of the incident rays are reflected onto the air/film interface, while the remainder are transmitted through the layer and reflected off the second film/substrate interface.  Because the reflected rays have different optical pathways, they are in phase shift and interfere with each other, causing the appearance of a colour.  When the thickness of the film changes (for example, if it is placed in contact with an enzyme capable of degrading the biopolymer), the colour is modulated.

Rapid, simple and sensitive detection
The first step in this test thus consists in applying the nanocomponents in successive coats so as to closely control the thickness of the layers and hence the colour, and to modulate the sensitivity of the device.  The film is then placed in contact with the test solution for a few minutes, after which it is rinsed and dried.  Immediately, and without the need for reading apparatus, the verdict is clear: if the colour is modified, enzymatic activity has indeed occurred.  
Another not inconsiderable advantage of this method is its sensitivity.  Compared with a standard colorimetric technique, the detector thus developed is 50 times more sensitive in the case of cellulose/xyloglucan layers and up to 200 times more sensitive with other biopolymers tested (pectins, xylans, proteins, etc.).

Towards new, high-throughput screening tests 
The aim is now to work in collaboration with teams specialised in microsystems in order to develop miniaturisation of the devices and their inclusion in 96-well test plates, the format most frequently used for the automated screening of genomic databanks and compatible with all commercial systems.  Use of this method will enable the rapid and sensitive screening of the numerous cell banks that are growing up, notably because of the development of metagenomics and the challenges linked to white biotechnologies.

Contact(s)
Scientific contact(s):

Associated Division(s):
Science for Food and Bioproduct Engineering
Associated Centre(s):
Pays de la Loire

Find out more

Patent: Méthode de détection d’activités enzymatiques hydrolytiques sans marquage à base de couches réflectives de biopolymères. Carole Cerclier, Bernard Cathala. Patent No. FR 1055529 dated 7 July 2010
This device is the subject of an ANR programme which started in 2011-2012 under the acronym of Reflex. It involves the Institut d'Electronique de Microélectronique et de Nanotechologie (IENM) in Lille, the LISBP in Toulouse (notably through the enzyme screening platform ICEO) and the INRA BIA Research Unit in Nantes (coordinator).

  • Photonic structures in biology, Vukusic, P.; Sambles, J. R. Nature, (424), (6950), 852-855, 2003.
  • Elaboration of Spin-coated Cellulose-Xyloglucan Multilayered Thin Films, Carole Cerclier, Fabrice Cousin, Hervé Bizot, Céline Moreau and Bernard Cathala, Langmuir, 26 (22), pp 17248–1725, 2010.
  • Cerclier C., Lack-Guyomard A., Moreau C., Cousin F., Beury N., Bonnin E, Jean B., Cathala B. (2011) Coloured Semi-reflective Thin Films for Biomass-hydrolyzing Enzyme Detection. Advanced Materials 23: 3791–3795
films nanométriques sont obtenus par dépôts successifs de cellulose et de xyloglucane.

On the subject of

Illustration: the nanometric films are obtained using successive coats of cellulose and xyloglucan. The growth of the film is linear, depending on the number of applications (n) and a colour appears after a few cycles.  The layers are subjected to the action of cellulase solutions endowed with different enzymatic activities.  After incubation for a few minutes, followed by washing and drying, the colour of the film is modified or disappears totally.