Highly Sensitive Nanoparticle-based Multifunctional Biosensor for Antigen Detection

Precise and selective positioning of nanoparticles gives rise to many applications where assembly of nano building blocks with different biological or chemical functionalization is necessary. One remarkable application is the simultaneous early detection of multiple biomarkers in the field of miniaturized multiplex biosensors. To enable multiplex detection of antigens, nanoparticles with various antibody coatings can be selectively assembled in trenches on different regions on a biochip so that they bind selectively to the specific antigen of interest. The presented work utilizes electric field assisted assembly techniques to assemble nanoparticles with various surface functionalization and coatings. Nanoparticles are assembled into pre-fabricated via and trench patterns generated on a PMMA coated gold surface, using electron-beam lithography. Two techniques have been developed for selective assembly of nanoparticles: sequential size-selective directed assembly and sequential site-selective assembly. Both selective assembly techniques provide fast and reproducible assembly over large areas while achieving high yield. The sequential size-selective assembly is a template-assisted technique where the selectivity is achieved by controlling the size of the nanopatterns and the size of the nanoparticles. The possibility of particle detachment and the factors affecting the sorting efficiency for this technique is studied. We show that a complete sorting can be achieved when the size of the vias is close to the diameter of the nanoparticles and the size distribution of the chosen nanoparticles do not overlap. In the site-selective assembly, the selectivity is achieved by having electrically isolated sites (regions) on the same chip. Electrophoresis is performed for each region in a step by step process. Selective assembly results, for up to four nanoparticles with various coating/functionalization are presented using the site-selective assembly technique. We use the electrophoresis technique to assemble the cancer specific anti-PSA, mAb-2C5 and CEA coated nanoparticles to show that the nanoparticle-based biochip can successfully measure low concentrations of various antigen. The principle of operation of these biosensors is the fluorescence based ELISA. Testing results of the nanoparticle-based biochips indicate very high specificity and the detection limit 200 times smaller than the commercially available devices for antigen detection, laying the foundation for early detection of various diseases. The optimized assembly of antibody coated particles and selective assembly techniques introduced in this work provide the necessary tools for fabricating a miniaturized nanoparticle-based in-vivo multiplex biosensor. The antigen detection results show the great potential for early detection of various diseases using the fabricated in-vivo device.