A CONVENIENT APPROACH TO GLYCOAMPHIPHILES AS ACTIVE LAYERS OF LCD-BASED BIOSENSORS – SWEET-DISPLAY

The SWEET-DISPLAY project uses a multi-step approach to develop a new microbe detection technology based on innovative materials called liquid crystals. These materials can react visually to the presence of certain molecules, which could eventually lead to the creation of quick, simple, and effective screening tests.

The first stage (WP1) involves manufacturing new molecules called glycoamphiphiles (GAs). These are hybrid molecules with a “sweet” part (the head) that can bind to certain proteins found on microbes (lectins) and a “fatty” part (the tail) that allows them to attach to the surface of liquid crystals. Two complementary methods are being explored to create these GAs from readily available natural sugars, without complex chemical protection steps. These methods use specific chemical reactions developed in Dr. Sami Halila's laboratory and make it possible to obtain a wide variety of GAs with different properties by modifying the shape or length of their hydrophobic (fatty) part.

The second stage (WP2) involves testing these GAs in devices using liquid crystals. Two types of sensors are currently being developed. The first is based on liquid crystals known as “nematic” crystals, which are already well known in screens. These crystals change their appearance under a microscope when they detect an interaction between a GA and a microbial lectin. The aim is to verify whether the GAs organize themselves well on the surface of the liquid crystal and whether they react specifically to certain lectins, such as those produced by the bacterium Pseudomonas aeruginosa. The sensitivity, accuracy, and stability of the signal obtained will then be measured.

In a second stage, a second-generation sensor is being developed. This uses another type of liquid crystal, known as cholesteric, which can change color when it detects an interaction with a microbe. This system, inspired by the colors observed in certain insects and shellfish, would allow for even simpler visual reading, without a microscope, by detecting color changes visible to the naked eye or measurable by a small optical device.

Finally, in the third stage (WP3), the microbial lectins to be detected are produced in the laboratory. Four lectins are being studied, originating from human pathogens (P. aeruginosa, B. ambifaria) or plant pathogens (R. solanacearum). The ability of GAs to bind to these lectins in solution is then measured in order to better understand their effectiveness before testing them in biosensors.

By combining chemistry, biotechnology, and materials science, SWEET-DISPLAY paves the way for smart sensors for rapid microbe detection that can be used in hospitals, agriculture, or even at home.

The ANR SWEET-DISPLAY project aimed to develop new molecules and biosensors capable of detecting adhesion proteins called lectins, produced by certain pathogenic bacteria such as Pseudomonas aeruginosa. This research aims to pave the way for new rapid detection strategies in the field of health.

The researchers developed an innovative and rapid method for manufacturing hybrid molecules, combining a “sugar” part and a “fat” part (called glycoamphiphiles). These molecules have the particularity of spontaneously self-assembling in water to form nanoparticles.

The study showed that the shape and stability of these nano-objects depend heavily on how the molecules are constructed. These structures are robust enough to withstand different environments (pH, temperature), making them interesting for applications such as biosensors.

A scientific article on these results was published in 2024 in Bioconjugate Chemistry.

The second step was to create a biosensor based on liquid crystals, similar to those used in television screens. The principle is ingenious: when the liquid crystals are properly aligned, the image is dark; but if a lectin binds to the sugars placed at the interface, the organization of the crystals is disrupted and a “colored” light appears.

The researchers successfully developed an initial prototype capable of specifically detecting LecA lectin, produced by P. aeruginosa, at very low concentrations. This is encouraging proof of concept, even though the device still needs to be optimized. However, the design of a “second-generation” biosensor could not be completed within the project timeframe.

At the same time, the team produced and purified four lectins from pathogenic bacteria. ITC (isothermal titration calorimetry) analyses showed that glycoamphiphiles and their nano-assemblies interacted well with these proteins, with a measurable affinity in the micromolar range. These results confirm the relevance of the new molecules as detection elements.

Project summary:

In summary, SWEET-DIAPLY has made it possible to i) develop a new family of sugar-lipid hybrid molecules capable of organizing themselves into stable nanostructures; ii) demonstrate their specific interaction with bacterial lectins; iii) create a first prototype biosensor using liquid crystals to detect these interactions.

Although not all objectives were achieved, the project has opened up promising prospects for the simple and rapid detection of bacterial markers. This work could ultimately contribute to the development of innovative tools for medical diagnosis.

The SWEET-DISPLAY project has led to the development of new hybrid molecules (glycoamphiphiles) and an initial biosensor prototype. These results open up numerous avenues for both fundamental research and practical applications in healthcare, biotechnology, and even cosmetics.

1. Improving and diversifying new molecules

The method developed makes it easy to manufacture glycoamphiphiles by combining different “sugar” and “fat” components. The next step is to expand this library of molecules, for example by using different sugars (galactose, mannose, fucose, etc.) to target other proteins of interest, particularly lectins produced by pathogenic bacteria.

These new combinations could also be explored in other areas: for example, as nanovectors capable of transporting and delivering drugs in a targeted manner.

2. Towards more efficient biosensors

The first-generation biosensor has shown that it is possible to specifically detect a lectin from Pseudomonas aeruginosa. However, several steps are necessary to make it a reliable tool: improving its sensitivity, better defining its detection threshold, and optimizing its stability over time.

The researchers also want to resume development of a second-generation biosensor, with more sophisticated liquid crystals or hybrid supports (such as polymer films). In the longer term, integrating these systems into miniaturized microfluidic devices could lead to rapid, automated testing.

3. Gaining an in-depth understanding of molecular interactions

Initial results have confirmed that glycoamphiphiles interact specifically with certain bacterial lectins. The next steps will aim to gain a better understanding of these interactions at the atomic level, using techniques such as NMR, crystallography, and computer modeling.

It will also be interesting to expand the range of proteins tested and examine the behavior of molecules in more complex biological environments that are closer to reality. Ultimately, this research could pave the way for systems capable of directly blocking or trapping bacteria, an approach known as “anti-adhesion.”

4. Industrial applications and commercialization

The advances made by SWEET-DISPLAY are not limited to the academic field. They could find applications in:

i) in medical diagnostics, with portable, rapid biosensors;

ii) in pharmacy, to screen proteins and test new therapeutic strategies;

iii) in cosmetics, as stabilizing agents or components of innovative formulations.

Finally, the methods developed could lead to patents, opening the door to partnerships with manufacturers and concrete commercialization of the research.

Glycoconjugates, defined here as carbohydrates attached to a lipid or a protein, are ubiquitous in Nature. They are present on all cell membranes through a dense coating named Glycocalyx. They play critical roles in a wide variety of biological and pathological processes acting as signaling, recognition, and bacterial adhesion, etc. Consequently, major scientific and biotechnological interests in accessing glycoconjugates derive from the promise to use them as probes for biological research, as well as lead compounds for developing drugs, vaccines, and diagnostic tools. These endeavors are however complicated by a lack of general methods for the straightforward preparation of these key-enabling carbohydrate derivatives. This is a major scientific challenge for glycochemists worldwide.

The transdisciplinary (Chemistry/Biology/Nano-science/technology) SWEET-DISPLAY project aims at developing a new modular chemical platform based on barbituric acid, allowing direct access to a wide range of glyco-amphiphiles (GAs) or glycolipids analogues through Knoevenagel condensation on protecting group-free carbohydrates. These GAs will act as active recognition layers of pathogenic lectins in liquid crystal (LC) biosensors. In fact, Prof. N.L. Abbott’s group has pioneered, and successfully developed over the past two decades, the smart use of LCs to transduce and amplify molecular events at an aqueous/LC interface; allowing their detection though optical signals and images visible to the naked eye. This LC biosensor technology is capable of delivering a simple, high-sensitivity, and label-free detection without the requirement of complex instrumentations, making it well-suited for the primary screening assay of analytes performed away from central laboratories. LC biosensors have already been designed with many amphiphilic species (e.g. surfactants and lipids) to detect a hand full of biological analytes (e.g. including proteins, nucleic acids, viruses, endotoxins, and cells to name few) but never challenged till date for probing carbohydrate/pathogenic lectin (a carbohydrate-binding protein) interactions, pointing out toward the inherently innovative application of this Public Collaborative Research (PRC) exploratory project. Lectins such as LecA (galactophilic) and LecB (fucophilic) from Pseudomonas aeruginosa, a bacterium that have become a real concern in hospital-acquired infections will be first covered. Other biotargets of interests are RSL (fucophilic) from the plant pathogen Ralstonia solanacearum that leads to lethal wilt in many agricultural crops or its homolog BambL from human pathogen Burkholderia ambifaria that was identified in clinical isolates from cystic fibrosis patients.

SWEET-DISPLAY is a low TRL (1-3) PRC project grounded on the unification of cross-fertilizing knowledge and complementary expertise of two internationally recognized labs uniting for the first time under a PRC: CERMAV, in glycosciences, and SyMMES, in functional liquid crystals. Backed on preliminaries results, its core novelty lies in an original access to a large range of GAs characterized by hydrophobic tails that will penetrate into the hydrophobic LC phase while the hydrophilic carbohydrate heads will remain exposed to the aqueous phase, defining a functional self-assembled monolayer at the LC/GA/water interface. Pathogenic lectins will cause local disruptions of this sensing interface that will propagate through the bulk of LCs providing an optical readout through polarizing microscopy images (with nematic LCs-Generation 1). A 2nd generation, even more appealing and easy to implement, enabling a color-indicating assay (with cholesteric (chiral nematic LCs-Generation 2) will be proposed and will rely on the sensitivity of Bragg reflections (induced by the helical self-assembly of cholesteric LCs) to temperature and pathogen concentration when sense with a fiber optic-based UV-Vis-NIR spectrophotometer.