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.
The early detection of biological warfare (BW) agents before any symptoms are present is critical for saving lives and reducing cost of therapy. Protein expression in T-cells represents one of the earliest detectable cellular signaling events to occur in response to the exposure to various toxins or BW agents. In order to fully understand a cellular response to a particular BW agent, it is often necessary to monitor the expression of specific proteins. Therefore, we have developed a novel class of surface enhanced Raman scattering (SERS) immuno-nanosensors for the real-time monitoring of protein expression within individual living cells.In this work, we have developed and optimized novel nanosphere-based silver coated SERS nanosensors for the detection of proteins at cellular levels. SERS nanosensors were optimized in terms of nanosphere size, silver coating methods, number of silver layers, antibody binding and affinity. These nanosensors are capable of being inserted into individual cells and non-invasively positioned to the sub-cellular location of interest using optical tweezers. They were constructed from monodisperse silica nanospheres. These nanospheres were condensed from tetraalkoxysilanes in an alcoholic solution of water and ammonia. Accurate control of the silica nanospheres' diameter was achieved by varying the reaction conditions. Nanosphere-based SERS immuno-nanosensors were then prepared by depositing multiple layers of silver on silica spheres, followed by binding of the antibody of interest to the silver. In binding the antibodies, different cross linker agents were characterized and compared. On one end, each of these cross linker agents 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 improve sensitivity of these nanosensors, optimal silver surface coverage with crosslinkers was determined. Following binding of antibodies, evaluation of the nanosensors was performed by monitoring the SERS spectra of the nanosensors prior to and following exposure to the antigen of interest. These results showed reproducible differences in the SERS spectra upon exposure to the antigens confirming their ability to monitor trace amounts of antigen. In particular, these SERS-based nanosensors were shown successfully detect human insulin at trace levels.
We report template-free electrochemical deposition method for preparing ZnO nanostructures arrays on indium tin oxides (ITO) glass substrate. Multiform ZnO nanostructures, such as nanotubes, nanorods with tower-like tips, cone-like tips and groove-like tips, are controllably synthesized at 60 °C, which is lower compared with the prepared temperatures of reported works. The results of XRD indicate the wurtzite ZnO nanostructures are single-crystalline and grow along the c-axis perpendicularly on the substrate. These findings have potential for the growth of high-quality ZnO nanostructures arrays and device applications.
Gold-based surface-enhanced Raman scattering (SERS) beacons have been developed, which represent a simple, biocompatible and rapid means of performing multiplexed DNA sequence detection in a non-arrayed format. These SERS beacons consist of a simple stem-loop oligonucleotide probe in its native form with one end attached to a SERS active dye molecule and the other to a gold nanoparticle, approximately 50 nm in diameter. The probe sequence is designed to achieve a stem-loop structure, with the loop portion complementary to the target sequence, similar to fluorescent molecular beacons. In the absence of the target DNA sequence, the SERS signal of the associated dye molecule is detected, representing the "ON" state of the probe. When the target sequence is hybridized to the probe, which results in an open conformation, its respective reporter dye is separated from the gold nanoparticle, producing diminished SERS signal. In this paper, the fabrication and characterization of these SERS beacons is described. We also demonstrate selective hybridization of a target sequence to one beacon in a mixture, revealing their potential for use in a multiplexed fashion.
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