Li-Lin Tay

Single‐Domain Antibody‐Conjugated Nanoaggregate‐Embedded Beads for Targeted Detection of Pathogenic Bacteria

The rapid screening of pathogenic bacteria remains a key issue in the diagnosis of infectious diseases, food safety, and public health assurance. In particular, the emergence of drug-resistant bacteria presents great challenges to the health care sector. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for one of the better-known hospital-acquired infections. S. aureus is a common pathogen that can colonize various areas of the human anatomy. Healthy individuals may carry MRSA asymptomatically, but patients with a compromised immune system are at a greater risk of symptomatic secondary infection. Due to the drug-resistant nature of S. aureus, preventive measures, such as routine patient screening, remain the most effective way to control the spread of this bacterium in clinical environments. In the clinical setting, routine analysis of pathogenic bacteria typically involves biochemical characterization of cultured microorganisms taken from contaminated sources. Standardized procedures are time consuming, which thus highlights the need for a rapid and targeted detection methodology. Advances in nanotechnology and biotechnology offer new possibilities for the rapid screening of harmful microorganisms. Surface-enhanced Raman scattering (SERS) has been demonstrated to achieve ultra-high sensitivity detection in many bioanalytical assays. Herein, we combine the high sensitivity of a newly developed SERS nanoprobe with the high specificity of single-domain antibody (sdAb) to achieve the targeted detection of a single bacterial pathogen, S. aureus. The ability of metallic nanostructures to localized surface plasmon resonance (LSPR) under appropriate electromagnetic field excitation is largely responsible for the SERS effect. The LSPR is strongly dependent on the size and shape of the nanostructures. It has been demonstrated that extremely high SERS enhancement can be achieved when nanostructures are closely spaced, which allows their LSPR to couple. At the optimal interparticle spacing, LSPR coupling results in the capacitive enhancement of the Raman effect for molecules located between particles in the SERS “hot sites”, which thus enables the detection of very few molecules under optimal excitation conditions. A specially designed SERS nanoprobe called a nanoaggregateembedded bead (NAEB) was fabricated with this optimization in mind. NAEBs are fabricated by controlled formation of small Au nanoparticle (NP) aggregates that are subsequently encapsulated in a protective silica shell (Scheme 1a), and fully utilize the advantage of LSPR coupling of a small NP aggregate; as a result each nanosized bead is an ultrahigh sensitivity SERS nanoprobe. Raman reporter molecules (R6G) were incorporated into the nanoaggregate during the formation process to give each NAEB a unique Raman signature. Compared with other SERS-based bioanalytical applications that utilize Ag or Au NPs, NAEBs have the added advantage of stability. Without the protective silica shell, even passivated Au or Ag NPs immersed in biological buffers are prone to parasitic signals from adsorption of the molecules in the biological fluids or loss of signals due to the desorption of the Raman reporter molecule. This problem was recognized by the groups of Liz-Marzan, Natan, Nie, and Brown, who pioneered the work on encapsulating Au NPs with a protective silica shell to improve their stability. In these earlier works, encapsulation was done without deliberate aggregation, which resulted in a relatively low [a] P.-J. Huang, Prof. L.-K. Chau Department of Chemistry and Biochemistry National Chung Cheng University 168 University Road Min-Hsiung, Chia-Yi (Taiwan) Fax: (+886)5-2721040 E-mail : chelkc@ccu.edu.tw [b] Dr. L.-L. Tay, Dr. J. Tanha, S. Ryan Institute for Microstructural Sciences and Institute for Biological Sciences National Research Council Canada Ottawa, ON K1A 0R6 (Canada) Fax: (+1) 952-6337 E-mail : lilin.tay@nrc-cnrc.gc.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901397.

Nanoscale aggregation of cellular beta2-adrenergic receptors measured by plasmonic interactions of functionalized nanoparticles.

Adrenergic signaling that controls the contraction of cardiac myocyte cells and the beating of the mammalian heart is initiated by ligand binding to adrenergic receptors contained in nanoscale multiprotein complexes at the cellular membrane. Here we demonstrate that the surface-enhanced Raman scattering (SERS) of functionalized silver nanoparticles can be used to report on the receptor aggregation state of specifically label beta(2)-adrenergic receptors on mouse cardiac myocyte cells. Furthermore, multimodal imaging including Raman, Rayleigh scattering, scanning electron microscopy, and luminescence imaging was combined to fully characterize the beta(2)-adrenergic receptor-mediated aggregation of silver nanoparticles on the membrane of cardiac myocytes. Scanning electron microscopy analysis reveals distinct SERS active clusters of between 10 and 70 nanoparticles per signaling domain from ultra-high-resolution images of beta(2)-adrenergic receptor clusters on the cellular membrane. These techniques can be generally applied to study the aggregation of other cell surface receptors and explore their distribution on cell surfaces.

Single-Domain Antibody-Nanoparticles: Promising Architectures for Increased Staphylococcus aureus Detection Specificity and Sensitivity

Because antibodies are highly target-specific and nanoparticles possess diverse, material-dependent properties that can be exploited in order to label and potentially identify biomolecules, the development of antibody-nanoparticle conjugates (nanoconjugates) has huge potential in biodiagnostics. Here, we describe a novel superparamagnetic nanoconjugate, one whose recognition component is a single-domain antibody. It is highly active toward its target Staphylococcus aureus, displays long shelf life, lacks cross-reactivity inherent to traditional homologue whole antibodies, and captures a few dozen S. aureus cells in a mixed cell population with ~100% efficiency and specificity. We ascribe the excellent performance of our nanoconjugate to its single-domain antibody component and recommend it as a general purpose recognition element.

Fourier-transform infrared and photoluminescence spectroscopies of self-assembled monolayers of long-chain thiols on (001) GaAs

Self-assembled monolayers (SAMs) of various thiols have shown the potential to protect freshly fabricated or chemically cleaned GaAs surfaces from oxidization, adsorption of foreign atoms, and∕or surface defect formation. We have employed an attenuated total reflection Fourier-transform infrared spectroscopic technique to investigate the process of formation of long-chain thiols, comprising ten or more methylene chains, on the surface of (001) GaAs. A strong infrared (IR) signal was measured for all the investigated GaAs-thiol interfaces. Varying the type of terminal groups, from hydrophilic to hydrophobic, significantly changes the IR intensity of the methylene stretching vibration, indicating different methylene chain orientation in SAMs. Consequently, these SAMs exhibited different passivation performance to the (001) GaAs surface as judged by the intensity of the GaAs-related photoluminescence signal.

Multiple surface plasmon resonances and near-infrared field enhancement of gold nanowells.

Arrays of Au nanowells (NWs) were fabricated by electron-beam lithography (EBL) and characterized by surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS). It is revealed that these Au NW arrays exhibit multiple SP resonances that can be tuned by adjusting the geometrical characteristics of the NWs. SERS activity of Au NWs was confirmed for a range of excitation wavelengths and a number of model compounds including rhodamine 6G (R6G), phthalazine, and single-stranded oligonucleotides. According to numerical simulations based on the discrete dipole approximation (DDA), SERS enhancement originates from high electromagnetic fields (hot spots) localized both inside and outside individual NWs. In addition, far-field intercoupling effects between NWs have been observed experimentally in arrays with subwavelength pitch sizes. We show that the SERS enhancement factors can also be tuned and optimized by adjusting the geometry of NWs.

Suppression of interfacial reaction for HfO2 on silicon by pre-CF4 plasma treatment

In this letter, the effects of pre-CF4 plasma treatment on Si for sputtered HfO2 gate dielectrics are investigated. The significant fluorine was incorporated at the HfO2∕Si substrate interface for a sample with the CF4 plasma pretreatment. The Hf silicide was suppressed and Hf–F bonding was observed for the CF4 plasma pretreated sample. Compared with the as-deposited sample, the effective oxide thickness was much reduced for the pre-CF4 plasma treated sample due to the elimination of the interfacial layer between HfO2 and Si substrate. These improved characteristics of the HfO2 gate dielectrics can be explained in terms of the fluorine atoms blocking oxygen diffusion through the HfO2 film into the Si substrate.

Raman and transmission electron microscopy study of disordered silicon grown by molecular beam epitaxy

Silicon films were deposited by molecular beam epitaxy onto crystalline silicon (c-Si) and native oxide on c-Si (001) substrates at temperatures ranging from 98 to 572 °C. Raman spectroscopy of these films showed that both the short-range disorder and intermediate-range disorder decreases as the deposition temperature increases. The onset of a phase transition in the amorphous Si films can be effectively identified by the appearance of the polycrystalline and crystalline Si Raman bands, which allowed quantification of the crystalline volume fractions present. Both the transmission electron microscopy and Raman results confirmed that films grown on the amorphous substrates at temperatures less than 414 °C are entirely amorphous, but exhibit c-Si features at higher temperatures. Films grown on c-Si substrates exhibit a characteristic limiting thickness for epitaxy and the transformation of the resulting upper amorphous layer into crystalline form takes place at a much lower temperature (∼290 °C) than for th...

Influence of growth temperature on order within silicon films grown by ultrahigh-vacuum evaporation on silica

We study the role that the growth temperature plays in determining the amount of order present within silicon films deposited on fused silica substrates through ultrahigh-vacuum evaporation at growth temperatures ranging from 98 to 572°C. Through measurements of the Raman and optical absorption spectra, we quantitatively determine how the growth temperature influences the order present within 11 such films. We employ three disparate measures of order for the purposes of this study: the breadth of the transverse-optic phonon Raman peak, this being related to the amount of short-range order present; the area under the transverse-acoustic Raman peak divided by the area under the corresponding transverse-optic peak, this being related to the amount of intermediate-range order present; and the breadth of the optical absorption tail, which is a general measure of the overall amount of order present. All three measures of order indicate a dramatic increase in the amount of order present for growth temperatures a...

In situ monitoring of protein adsorption on functionalized porous Si surfaces

In situ monitoring of the photoluminescence (PL) of undecylenic acid functionalized porous silicon (PS) was carried out in bovine serum albumin (BSA) solution. Two factors dictated the PL behavior during the protein incubation process. A PL redshift was observed within a few hours of PS incubation in BSA solution followed by an overall gradual blueshift of the PL energy. Correlating this result with Fourier transform infrared (FTIR) spectroscopy performed before and after BSA incubation as well as with the PL and FTIR studies performed in parallel on planar Si, the redshift was attributed to the adsorption (or incorporation) of protein in the porous matrix while the blueshift was assigned to surface oxidation due to the erosion of Si nanocrystallites in the PS matrix. Understanding the protein incorporation in the PS matrix has important implications in utilizing the PL as an optical transduction method in a PS-based biosensor device.

Synthesis of surface enhanced Raman scattering active magnetic nanoparticles for cell labeling and sorting

Here we report the synthesis of the magnetic nanocomposite nanoparticles with Fe3O4 core and silver shell for cell imaging and separation. When the magnetic nanoparticles are decorated with surface enhanced Raman scattering (SERS) active molecules, they can be used for cell separation with unique optical signature. In this experiment, commercially available superparamagnetic nanoparticles (fluidMAG) with 50 nm diameter were used as the core. The shell layer was produced by the reduction of the silver salts. As a result of the reduction, nanocomposite magnetic nanoparticles with 60 nm diameter were obtained. To create unique SERS patterns for multiplexing, the surfaces of the nanoparticles were further modified with chloro-, bromo-, or fluorobenzenethiol. When these nanoparticles were incubated with 3T3 cells, it was found that the nanoparticles were located around the nucleus in the cytoplasm.