Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.

Biosensor-based detection provides a rapid and low-cost alternative to conventional analytical methods for revealing the presence of the contaminants in water as well as solid matrices. Although important to be detected, small analytes (few hundreds of Daltons) are an issue in biosensing since the signal they induce in the transducer, and specifically in a Quartz-Crystal Microbalance, is undetectable. A pesticide like parathion (M = 292 Da) is a typical example of contaminant for which a signal amplification procedure is desirable. The ballasting of the analyte by gold nanoparticles has been already applied to heavy target as proteins or bacteria to improve the limit of detection. In this paper, we extend the application of such a method to small analytes by showing that once the working surface of a Quartz-Crystal Microbalance (QCM) has been properly functionalized, a limit of detection lower than 1 ppb is reached for parathion. The effective surface functionalization is achieved by immobilizing antibodies upright oriented on the QCM gold surface by a simple photochemical technique (Photonic Immobilization Technique, PIT) based on the UV irradiation of the antibodies, whereas a simple protocol provided by the manufacturer is applied to functionalize the gold nanoparticles. Thus, in a non-competitive approach, the small analyte is made detectable by weighing it down through a “sandwich protocol” with a second antibody tethered to heavy gold nanoparticles. The immunosensor has been proved to be effective against the parathion while showing no cross reaction when a mixture of compounds very similar to parathion is analyzed. The immunosensor described in this paper can be easily applied to any small molecule for which polyclonal antibodies are available since both the functionalization procedure of the QCM probe surface and gold nanoparticle can be applied to any IgG, thereby making our device of general application in terms of target analyte.