[nanoPost] A top-down approach for designing optical nano-biosensors and biochips “on-demand”

Research Centre Italy

There is a substantial word-wide effort to develop non-invasive and minimally invasive methods for frequent and/or continuous monitoring of analytes of clinical, food and environment interests. In addition, high attention is also focused to design highly sensitive and specific sensors for homeland security.

A wide variety of methods potentially useful to measure the binding of analytes have been tested, including optical rotation, near-infrared absorbance, Raman scattering, as well as the design and synthesis of many chemical fluorescence probes.

 
Fluorescence detection is the dominant analytical tool in medical testing, biotechnology and drug discovery. The use of proteins and enzymes as specific probes for designing sensors for biochemical analytes has become a priority of the modern biotechnology.

A powerful set of tools based on the interactions of proteins with ligands such as ions, other macromolecules, and metabolites are being used to explore the molecular bases of life functions. Since proteins mediate with high specificity almost all the interactions and reactions in cells, they are well suited to act as the most important components of biosensors once it is possible to monitor the interactions with their ligands.

The widespread use of proteins as sensors depends on protocols to enhance stability, but an alternative method is to use naturally thermostable enzymes or proteins isolated from extremophiles. These macromolecules have intrinsically stable structural features, and they can be considered as ideal probe for the construction of advanced biosensors.

However, the use of enzymes as biosensors presents the disadvantage of the substrate consumption, which is a disadvantage in the design of implantable clinical sensors or in biotechnology assays.

In our Lab we have addressed the concern of the substrate consumption by using coenzyme-depleted enzymes as fluorescence probes for the development of an innovative class of non-consuming substrate biosensors. Apo-enzymes are still able to bind the substrate, but not to transform it. Additionally, the binding of substrates may result in conformational changes which can be easily detected by fluorescence and/or polarization measurements. There is a wealth of knowledge on enzymes which transform numerous substances of biochemical interest. Hence, the possibility of using inactive apo-enzymes as reversible sensors greatly expands the range of biochemically relevant analytes which can be measured using proteins as sensors.  In addition, we have also developed a fluorescence platform of advanced methodologies that allows the study of protein-protein and/or ligand-protein interactions even at single molecule level. This capability in biophysical methodologies together with expertise in protein biochemistry, molecular genetics and microbiology allows the research team to challenge with cutting-edge biochemical questions as well as to rapidly answer to biotechnological requests from SMEs and public institutions.

In the last two years we have focused our attention in the biophysical characterization of proteins belonging to the ABC transporter system such as the glucose-binding protein, the glutamine-binding protein, the trehalose/maltose-binding protein, the maltose-binding protein from mesophilic and extremophilic sources. An advanced fluorescence characterization has been performed on the glucose-binding protein. In particular, we have investigated the role of calcium in the protein structure/stability/dynamics by time-resolved-fluorescence and phosphorescence. We have contributed to clarify the effect of the substrate binding on the stability and denaturation mechanism of the protein. Finally, we have also characterized the protein denaturation mechanism from a thermodynamic point of view.  Mutant forms of this protein have been produced and characterized. We have also performed similar a characterization on the glutamine-binding protein, the trehalose-binding protein. This huge set of results obtained on the above mentioned biomolecules has allowed us to design  advanced fluorescence biosensors for sensing glucose, glutamine, trehalose, maltose. The developed biosensors have also been engineered at micro- and/or nano-scale indicating a roadmap for the realization of multi-arrays and “Lab-on-Chip” sensors.

An additional field of investigation of the research team has been the development of new assays and/or immunoassays  for food toxins and heavy metals. A competitive fluorescence immunoassay has been realized for detecting the patulin toxin in juices based on the production of specific antibodies obtained against a patulin derivative.

A new methodology for monitoring the presence of cadmium in drinking water as been developed by using a reversed-displacement protein-based fluorescence biosensor.

International and national patent applications have been filled for protecting the intellectual properties for the developed procedures.

In 2005-2006 an impressive number of publications on high-rank journals has been obtained by the research team.

Actually, the research team plays an important role in an excellence network of European labs for the physical-chemical characterization of biomolecules under severe conditions as well as it is a part of an European Consortium for the development of frontier methodologies for monitoring food safety and homeland security.