The development of selective, ecofriendly, low cost, stable and recycling biosensors for molecules and biomolecules detection in different environments such as atmosphere, food industry or in clinical analyses is strongly required. Among the most studied bio-components, enzymes play a leading role, being a class of biological recognition elements which are selective and sensitive. In this regard, the fabrication of enzyme based biosensors has received a huge interest in the last years. In particular many laccase based biosensors have been developed since this oxidoreductase enzyme is used in different fields due to its robustness and cheapness. In the fabrication process, enzyme immobilization strategies appear as a key factor to develop an efficient tool with appropriate performances. We are currently manufacturing new promising amperometric laccase-based biosensors with unprecedented reuse and storage capabilities by using the ambient electrospray deposition (ESD) methodology as immobilization technique. These new devices will be characterized electrochemically and the effects of metal nanoparticles deposition on the surfaces of commercial screen printed electrodes will be also investigated. Thanks to an interdisciplinary team, the main goal of this project is to extend the knowledge of these high resistant biosensors at the molecular level by using different approaches, microscopies and analytical tools to prove how the ESD immobilization technique is suited to manufacture high performance devices in term of storage and reuse.
Immobilization of enzymes on the transducer surface is one of the main challenges in the design of biosensors. In this regards, different methods have been tested to fabricate optical, electrochemical or gravimetric enzymatic biosensors. Immobilization is the critical point to achieve storage stability, high sensitivity, high selectivity, short response time and high reproducibility. Immobilized biomolecules have to maintain their structure, their function and their biological activity after being anchored to the surface and not to be desorbed during the use of the biosensor. Moreover, an ideal biosensor has to be stable for long-term application. There are different immobilization methods and each of them presents advantages and drawbacks:
1. Entrapment
2. Adsorption
3. Cross-linking
4. Covalent immobilization
5. Affinity
In the entrapment methodology the enzyme is incorporated within a gel or a polymer. The adsorption is the easiest method but the enzyme easily desorbs after deposition due to the weak bonds involved. More efficient, in term of duration of the biosensor, are covalent immobilization, affinity and cross-linking strategies, even if the latter causes a high enzyme activity loss. In the last years, the laccase based biosensors have been attracting a lot of interest particular at the industrial level. The laccase is one of the most robust biocatalysts due to extracellular nature and it is used in wide applications spanning from the environmental to pharmaceutical fields. It is also considered the most suitable "green catalyst" enzyme requiring only oxygen molecules as reactants and producing only water molecules as byproducts. However, the biocatalytic processes are usually affected by the life time of the enzyme, stability of biosensor that limits their use at an industrial scale. Physical or chemical approaches have tested in different laccase's immobilization. However, the final solution is still under investigation.
Recently, during the progress of the funded regional project "DESIR", we have fabricated new promising green friendly electrochemical amperometric laccase based biosensors, trough ESD methodology, that show an unprecedented reuse and storage performance.
The present project precisely aims to investigate the reasons of the very efficient immobilization of enzyme laccase from Trametes versicolor on carbon black and simple carbon screen printed electrodes. The goal is very ambitious and of huge interest since at industrial level the storage and stability of the biosensors are fundamental parameters and they are strictly connected to immobilization strategies. The ESD technology is based on ions soft landing on a surface at ambient pressure and temperature. [1] What happens to the molecules during the all process from ambient ESI to electrode is still a difficult task even if many approaches and strategies have been used to rationalize the events. The soft landing should guarantee deposition of the intact ions or the corresponding molecules at the surface, avoiding the destruction of the materials. The ions could be neutralized during the impact with the surface or stay ionic or react with the surface. Other possibilities are the chemical reactions in the gas phase during the electrospray process. These reactions could prepare the target material to be immobilized on different surfaces without pretreating the working electrode with other chemicals reducing in this way the biosensor fabrication time and realizing a more green friendly manufacturing. In our specific case the immobilization of laccase by ESD is verified with electrochemical analysis. The study of this immobilization process at the molecular level proposed in this project have fundamental basis in the fabrications of biosensors and could improve the knowledge of the chemical processes that could be used for the production of other type of sensors and biosensors. In literature there are few examples of enzyme based biosensor obtained with ESD and their characterization at the molecular level are scarce. Among the different methods described to immobilize enzymes, drop casting is one of the cheaper. However, this method does not produce stable and long lived biosensor and hence other more sophisticated procedures have been realized, and among them the electrospray ionization deposition mainly under vacuum was promising together with the ambient soft landing ESD more recently developed. In our specific case the laccase enzyme purchased from Sigma Adrich is probably a mixture of materials that could help the immobilization of laccase on the surface by trapping this enzyme in its natural environment maintaining its activity also after deposition. In this proposal all these aspects will be investigated with joint experimental techniques and rationalized at the molecular level to improve the knowledge of the immobilization methodology.
Reference
[1] A. K. Badu-Tawiah et al Anal. Chem. 2011, 83, 2648-2654