We have developed a novel class of gold multilayer, surface-enhanced Raman scattering (SERS) substrates that are capable of enhancing SERS signals by 15.3-fold over conventional gold film over nanostructure (GFON) SERS substrates, making them comparable in sensitivity to optimized silver film over nanostructure (SFON) substrates, while providing the long-term stability obtained from gold. They are fabricated by depositing 10 A thick silver oxide islands on conventional GFON substrates, followed by deposition of a second continuous gold layer. The silver oxide layer acts as a dielectric spacer between the two continuous gold films and produces significantly enhanced SERS signals, as compared to optimized single layer substrates of the same geometry or comparable substrates prepared by deposition of silver islands that are not oxidized. In addition to the enhanced sensitivity of these multilayer substrates, they also exhibit long SERS active shelf-lives (i.e., months), with no measurable degradation in SERS enhancement, and relative standard deviations in SERS enhancement of less than 5.2% across the substrate's surface.
This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.(Nanobiotechnology
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.
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