A novel surface enhanced Raman scattering (SERS)-based immuno-microwell array has been developed for multiplexed
detection of foodborne pathogenic bacteria. The immuno-microwell array was prepared by immobilizing the optical
addressable immunomagnetic beads (IMB) into the microwell array on one end of a fiber optic bundle. The IMBs, magnetic
beads coated with specific antibody to specific bacteria, were used for immunomagnetic separation (IMS) of corresponding
bacteria. The magnetic separation by the homemade magnetic separation system was evaluated in terms of the influences of
several important parameters including the beads concentration, the sample volume and the separation time. IMS separation
efficiency of the model bacteria E.coli O157:H7 was 63% in 3 minutes. The microwell array was fabricated on hydrofluoric
acid etched end of a fiber optic bundle containing 30,000 fiber elements. After being coated with silver, the microwell array
was used as a uniform SERS substrate with the relative standard deviation of the SERS enhancement across the microwell
array < 2% and the enhancement factor as high as 2.18 x 10<sup>7</sup>. The antibody modified microwell array was prepared for
bacteria immobilization into the microwell array, which was characterized by a sandwich immunoassay. To demonstrate the
potential of multiplexed SERS detection with the immuno-microwell array, the SERS spectra of different Raman dye labeled
magnetic beads as well as mixtures were measured on the mircrowell array. In bead mixture, different beads were identified by
the characteristic SERS bands of the corresponding Raman label.
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.
A multilayer surface-enhanced Raman scattering (SERS) substrate geometry providing
significantly greater SERS enhancements, longer active lifetimes, better reproducibility, and lower
detection limits for trace chemical analysis than traditional SERS substrates has been developed. We
have fabricated and characterized this novel class of multilayered metal film-based SERS substrates,
which are capable of enhancing SERS signals over an order of magnitude relative to conventional
metal film over nanostructure substrates. These multilayer enhanced metal film substrates are
fabricated by repeated vapor deposition of metal films over nanometer sized structures. Different
sizes of nanostructures were evaluated in order to obtain the optimal SERS enhancements.
Meanwhile, different dielectric coatings were fabricated between silver layers, and SERS
enhancements were evaluated for each type. Additionally, different metals, such as gold, were used
to further optimize the stability and reproducibility of these novel substrates. Silver oxide layers
produced at elevated temperatures were also investigated to accelerate the fabrication rate of these
multilayer substrates. Finally, this paper also discusses the application of these novel multilayer
substrates for trace detection of chemical agents and simulants.
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 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.