High sensitive sensing by effective immobilization of UV photo-activated antibodies

Abstract

Nowadays there is a strong interest in precise and reliable measurement tools suitable for quantifying physical, chemical, and biological properties. Biosensors face this problem by exploiting the intrinsic specificity provided by biological sensitive molecules to reveal the presence of the compound of interest. In particular, proteins like antibodies have a dominant role in biosensor development since they are selected by host immune system to efficiently bind foreign species including bacteria, viruses and toxins. Biomolecules involved in biosensing are usually characterized by a recognition site responsible for the selective detection of the analyte. This portion of the macromolecule has to be accessible when the sensitive element is coupled with the inorganic transducer, thus making surface functionalization a crucial phase of biosensor development. This issue strongly motivates the research of new immobilization and functionalization techniques allowing the control on both amount and orientation of the biomolecules thus resulting in better sensitivity and lower limit of detection. Conventional functionalization strategies are based on covalent and non-covalent interactions between the biological element and the surface of the transducer. Even if covalent approaches provide an effective immobilization of the biomolecules, these methods are laborious and time-consuming since several chemical treatments and purification steps are needed. In addition, the high toxicity of some chemicals and the complexity of the procedure require trained operators. On the other side, non-covalent immobilization is much easier to realize since it involves the spontaneous adsorption of the biomolecules onto the substrate. It is worth mentioning that in this case uncontrolled adsorption usually results in irregular layers and compromised recognition of the analyte due to steric hindrance of the binding sites. In addition, weak connections like van der Waals and hydrogen bonding interactions sometimes do not provide a stable immobilization onto the sensor surface. To face this issue, at the Physics Department of University of Naples Federico II, an all optical technique (PIT, Photonic Immobilization Technique) based on the interaction of ultrashort UV pulses with antibodies has been proposed as a simple and rapid approach capable to effectively functionalize the sensitive surface of a quartz crystal microbalance. In this thesis, PIT has been used to realize immunosensors for the detection of a group of analytes of practical interest. This functionalization technology provides an effective immobilization of antibodies onto the gold sensor surface upon activation of the protein sample through the selective photoreduction of the disulphide bridge in the triad cysteine-cysteine/tryptophan, a typical structural feature of the immunoglobulins. The absorption of ultrashort UV laser pulses required for this activation process does not affect the recognition properties of the antibodies. On the other side, the free thiol groups so produced interact with gold surface thus leading to the effective exposure of the sensitive portions of the protein, the so-called antigen binding sites, thus greatly improving the detection efficiency. The effects of this unconventional functionalization approach on immunoglobulins have been investigated by means of optical techniques, atomic force microscopy and the so-called Ellman's assay, a chemical method used to quantify the thiol groups in a protein sample. PIT based immunosensors have proven to be effective in the detection of small toxic molecules like parathion (pesticide) and patulin (micotoxin). The issue of revealing these light molecules using a microgravimetric transducer like a quartz crystal microbalance have been overcome by ballasting the analytes using two labelling procedures involving either bovine serum albumin or an antibody in a sandwich-type configuration. PIT has been used also to realize an immunosensor for the detection of gliadin, the principal responsible for the coeliac disease. In all these cases, both sensitivity and limit of detection (usually in nanomolar concentration range) result to be in line with the limits set by current regulations and comparable or even better than other techniques used to quantify these harmful molecules. These promising results make PIT a valuable functionalization method for technologies involving gold surfaces for sensing and detection purposes.