A critical aspect of the use of nanoprobes for intracellular studies in chemical and biological sensing involves a fundamental understanding of their uptake and trajectory in cells. In this study, we describe experiments using surface-enhanced Raman scattering (SERS) spectroscopy and mapping to track cellular uptake of plasmonics-active labeled nanoparticles. Three different Raman-active labels with positive, negative, and neutral charges were conjugated to silver colloidal nanoparticles with the aim of spatially and temporally profiling intracellular delivery and tracking of nanoprobes during uptake in single mammalian cells. 1-D Raman spectra and 2-D Raman mapping are used to identify and locate the probes via their SERS signal intensities. Because Raman spectroscopy is very specific for identification of chemical and molecular signatures, the development of functionalized plasmonics-active nanoprobes capable of exploring intracellular spaces and processes has the ability to provide specific information on the effects of biological and chemical pollutants in the intracellular environment. The results indicate that this technique will allow study of when, where, and how these substances affect cells and living organisms.
Surface-enhanced Raman scattering (SERS) was investigated for applications in the analysis of anthraquinone dyes used in works of art. Two SERS procedures were developed and evaluated with frequently used anthraquinone dyes, alizarin, carminic acid and lac dye. The first procedure involves the removal of a microscopic fragment containing alizarin from a painting, and a layer of silver nanoparticles was thermally evaporated directly on the fragment to induce SERS signal from alizarin. The applicability of this procedure for analyzing solid samples of color layer from paintings was discussed in detail. In the second procedure, a SERS-active substrate was prepared by spin-coating an alumina-nanoparticle layer onto a glass slide, followed by thermally evaporating a layer of silver nanoparticles on top of the alumina layer. Aliquots of dye solutions were delivered onto this substrate where intense SERS spectra characteristic of alizarin, carminic acid, and lac dye were obtained. The effects of two parameters, the concentration of the alumina suspension, and the thickness of the silver nanoparticle layer, on the performance of the Ag-Al<sub>2</sub>O<sub>3</sub> substrate were examined with alizarin as the model compound. Comparative studies with other common SERS substrates showed larger enhancement and improved reproducibility for the Ag-Al<sub>2</sub>O<sub>3</sub> substrate. The potential applicability of the Ag-Al<sub>2</sub>O<sub>3</sub> substrate for the analysis of real artifact objects was illustrated by the identification of alizarin extracted from a small piece of textile dyed with traditional methods and materials. The limit of detection for alizarin was estimated to be 7×10<sup>-15</sup> g from tests using solutions of known concentration.
This paper describes a compact, self-contained, cost effective, and portable Raman Integrated Tunable Sensor (RAMiTs) for screening a wide variety of chemical and biological agents for homeland defense applications. The instrument is a fully-integrated, tunable, "point-and-shoot" Raman monitor based on solid-state acousto-optic tunable filter (AOTF) technology. It can provide direct identification and quantitative analysis of chemical and biological samples in a few seconds under field conditions. It also consists of a 830-nm diode laser for excitation, and an avalanche photodiode for detection. Evaluation of this instrument has been performed by analyzing several standard samples and comparing the results those obtained using a conventional Raman system. In addition to system evaluation, this paper will also discuss potential applications of the RAMiTs for detection of chemical and biological warfare agents.
Nanoparticles are increasingly finding a wide application in the biological studies due to their unique physical and chemical properties. Colloidal nanoparticles are efficient substrate that exhibit surface-enhanced Raman scattering (SERS) phenomenon by enhancing the scattering cross-sections of conjugated Raman active molecules thus enabling highly sensitive biological probes. However, biological and medical applications would require nanoparticles to be conjugated to biomolecules. A universal approach for conjugation of mercarptoacetic acid-capped silver nanoparticles to biomolecules is described. The surface functionalized silver colloids were labeled with a Raman active dye and used for cellular imaging. We also described the use of silver nanoparticle to develop a new class of SERS nanoprobes for molecular recognition and detection of specific nucleic acid sequences.
This paper describes a self-contained, portable Raman instrument that has been developed for environmental and homeland defense applications. The instrument consists of a 830-nm diode laser for excitation, an acousto-optic tunable filter (AOTF) for wavelength discrimination, and an avalanche photodiode for detection. The primary component of this system is the AOTF and it has been selected based on its spectral range along with its high resolution, ~7.5 cm<sup>-1</sup>. Software has been developed in house using C programming language for controlling the instrument (i.e. the AOTF frequency, the signal acquisition, etc.). Evaluation of this instrument has been performed by analyzing several standard samples and comparing to a conventional Raman system. In addition to system evaluation, this paper will also discuss potential applications of this instrument to trace detection of hazardous chemicals using the Raman Integrated Tunable Sensor (RAMiTs) coupled with surface-enhance Raman scattering process.
An integrated multi-functional biochip based on integrated circuit complementary metal oxide semiconductor (CMOS) sensor array for use in medical diagnostics and pathogen detection has been described. The usefulness and potential of the biochip as a rapid, inexpensive screening tool for detection of bioenvironmental pathogens will be demonstrated. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air.
Surface-enhanced Raman scattering (SERS) spectra of chemical agent simulants such as dimethyl methylphonate (DMMP), pinacolyl methylphosphonate (PMP), diethyl phosphoramidate (DEPA), and 2-chloroethyl ethylsulfide (CEES), and biological agent simulants such as bacillus globigii (BG), erwinia herbicola (EH), and bacillus thuringiensis (BT) were obtained from silver oxide film-deposited substrates. Thin AgO films ranging in thickness from 50 nm to 250 nm were produced by chemical bath deposition onto glass slides. Further Raman intensity enhancements were noticed in UV irradiated surfaces due to photo-induced Ag nanocluster formation, which may provide a possible route to producing highly useful plasmonic sensors for the detection of chemical and biological agents upon visible light illumination.
We present the fabrication and characterization of silver island films with SiO<sub>2</sub> coatings for application in plasmonic sensors based on surface-enhanced fluorescence and Raman detection. The emission spectral properties of goat anti-mouse immunoglobulin (IgG) F(ab')<sub>2 </sub>labeled with one or two fluorescein residues were examined on substrates with metallic silver islands. The self-quenching of fluorescein emission was mostly eliminated when this antibody fragment was held 60-90 Å from the surface of metallic silver islands, and our preliminary experiments demonstrated an 8-fold emission intensity increase. Similar surfaces were also examined for surface-enhanced Raman analysis of Rhodamine 123, a potential drug for photodynamic therapy.