A New Tool for Biochemical Analysis: Combining Enzyme-Responsive Gels with LCD Detection

Cells secrete a range of enzymes that allow them to modify their environment and facilitate their growth. However, the presence in these secretions of particular enzymes that breakdown or modify surrounding connective tissue can be prognostic markers of disease, e.g. the enzyme hyaluronidase, which is a clinically reliable marker for bladder cancer. In this proposal we describe a new methodology for the detection of biochemical events; combining enzyme-sensitive gels with liquid crystal displays. This proposal is a conceptual jump that builds upon a unique enzyme-sensing system we have developed, which combined liquid crystal display (LCD) technology (as found in LCD calculator screens) with an easy-to-manufacture bioanalyte surface. Our new concept exploits the three-dimensional and biocompatible 'semi-wet' nature of enzyme-sensitive gels; the gel is a reservoir of bioanalyte that provides the LCDs with mechanical stability (giving robust, portable sensors), whilst also supporting the growth of disease-causing cells on the sensor. These transparent enzyme-responsive gels will lend mechanical strength to arrays of LCDs constructed on transparent plastic chips; using these chips will make the LCDs visible to the naked eye. In response to secretions from disease-causing cells, these devices will show patterns of light and dark LCDs, which will profile enzymatic activity and reveal cellular markers of disease. Our system will make the identification of disease-causing enzymes and cells much faster, easier and cheaper, having the advantage over existing techniques in that it will give rapid results outside the laboratory without needing complex instrumentation.

We will create a new type of biosensor by interfacing liquid crystal displays (LCDs) with enzyme-responsive hydrogels. These sensors will detect enzymes in complex mixtures or enzymes secreted by cells, e.g. providing biosensors for detecting metastatic cancer cells that are secreting marker matrix metalloproteinases. This system relies on the reorientation of 4'-cyano-4-pentyl biphenyl (5CB, a water immiscible thermotropic liquid crystal) at the interface with the aqueous analyte-containing hydrogel. 5CB orientation will be switched by the enzymatic digestion of the hydrogelator, and can be visualised as an optical dark/light transition when viewed sandwiched between crossed polarisers. Using the ambient light sources provides a power-free method of reporting on biochemical events. We recently used 5CB-based LCDs to create simple biosensors that discriminated between three different common proteases. The proposed research vastly expands the scope of this successful project and will provide a step change in analytical biochemistry, giving new LCD devices with higher sensitivity, increased portability and a readout visible to the naked eye; factors that will allow these LCD biosensors to be used outside the laboratory environment. Key to our analytical methodology will be the creation of a range of enzyme-responsive hydrogels, which will provide mechanical stability for the LCD device and allow the culture of living cells either within or on the surface of the hydrogel. These enzyme-responsive hydrogels will be interfaced with a sensing surface of LCD arrays, which have been fabricated on PDMS chips via soft lithography. In response to cellular secretions, these devices will give readouts of light/dark LCD patterns that profile enzymatic activity, for example from cells that are implicated in disease. Our system will make the identification of disease-causing enzymes and cells much faster, easier and cheaper without the need for complex instrumentation.