Nanosphere-based SERS immuno-sensors for protein analysis

Abstract

We have developed and optimized novel nanosphere-based silver coated SERS substrates for the detection of proteins. These SERS substrates were optimized for silver thickness, number of silver layers, and extent of silver oxidation between layers. Immuno-nanosensors capable of being inserted into individual cells and non-invasively positioned to the sub-cellular location of interest using optical tweezers were constructed from monodisperse silica nanospheres. Silica nanospheres ranging in diameter from 100 to 4500 nm were condensed from tetraalkoxysilanes in an alcoholic solution of water and ammonia. By varying the reaction conditions, accurate control of the silica nanospheres’ diameter was achieved. Silica sphere sizes were optimized for SERS signal response. Nanosphere-based SERS substrates were made by depositing multiple layers of silver on the nanospheres, followed by binding of the antibody of interest to the silver. In binding the antibodies, different crosslinkers were characterized and compared. On one end, each of these crosslinkers contained sulfur or isothiocyanate groups which bound to the silver surface, while the other end contained a carboxylic or primary amine group which reacted readily with the antibodies. In order to evaluate these substrates, SERS spectra of different proteins, such as insulin and interleukin-2 (IL-2), were obtained. By using silver, as the metal surface for SERS, red and near-infrared excitation wavelengths (i.e., 600-700 nm) can be used. Excitation in this range helps to avoid photodamage to cells and reduces any autofluorescence background. Evaluation of these SERS substrates was performed using a 10 mW HeNe laser, operating at 632.8 nm, in a collinear excitation/detection geometry. The SERS signals were filtered with a holographic notch filter, dispersed by 1/3 meter spectrometer and detected using an intensified charge coupled device (ICCD). This paper discusses the fabrication and optimization of these nanosensors, as well as their potential applications.