Advanced ELISA-like Biosensing Based on Ultralarge-Pore Silica Microbeads.

In this work, functionalized porous silica-based materials, widely used in the literature as drug and biomolecule nanocarriers, were innovatively used as an effective three-dimensional (3D) substrate for the development of a specific biomolecular assay showing great versatility in terms of detection performance. One-pot synthesis of ultralarge-pore silica microbeads was optimized to develop an enzyme-linked immunosorbent (ELISA)-like DNA detection assay. Cocondensation synthesis enabled introducing thiol functionalities into the silica framework while preserving both the high specific surface area (560 m2/g) and large pore size (17 nm average diameter), which are essential to guaranteeing high loading capability. Indeed, the bead-capturing ability was proved by developing an ELISA-like assay for the detection of short DNA sequences (≈20 bp), both in labeled and label-free configurations. In particular, the suppression of unspecific binding on the bead surface by testing two different blocking agents was a matter of interest. The detection performances were evaluated and compared to the ones obtained by following the same detection protocol on a standard flat surface (two-dimensional, 2D), which is most commonly used for this purpose. The bead-based assay showed a limit of detection two times lower than the flat-surface assay, confirming the promising capturing ability due to the larger active surface area. Furthermore, compared to traditional ELISA, the bead-based assay showed an intrinsic larger dynamic range that can be tailored depending on the final amount of beads used for the colorimetric quantification.

Polymeric 3D Printed Functional Microcantilevers for Biosensing Applications.

In this study, we show for the first time the production of mass-sensitive polymeric biosensors by 3D printing technology with intrinsic functionalities. We also demonstrate the feasibility of mass-sensitive biosensors in the form of microcantilever in a one-step printing process, using acrylic acid as functional comonomer for introducing a controlled amount of functional groups that can covalently immobilize the biomolecules onto the polymer. The effectiveness of the application of 3D printed microcantilevers as biosensors is then demonstrated with their implementation in a standard immunoassay protocol. This study shows how 3D microfabrication techniques, material characterization, and biosensor development could be combined to obtain an engineered polymeric microcantilever with intrinsic functionalities. The possibility of tuning the composition of the starting photocurable resin with the addition of functional agents, and consequently controlling the functionalities of the 3D printed devices, paves the way to a new class of mass-sensing microelectromechanical system devices with intrinsic properties.

Succinic anhydride functionalized microcantilevers for protein immobilization and quantification.

Microcantilever-based systems have been proposed as sensing platforms owing to their high sensitivity when used as mass sensors. The controlled immobilization on a surface of biomolecules used as recognition elements is fundamental in order to realize a highly specific and sensitive biosensor. Here, we introduce for the first time the application to a microcantilever-based system of a reliable chemical functionalization consisting of silanization with an aminosilane followed by a modification resulting in a carboxylated thin film. This chemical functionalization was tested for reproducibility of molecule deposition and for its protein grafting ability. Finally, this system was employed for the quantification of grafted proteins on the microcantilever surface. Moreover, a theoretical surface density of immobilized proteins estimated with bioinformatics tools was compared with the experimental surface density data, providing information about the orientation that the biomolecules assumed with respect to the sensing surface.