Nanostructure substrates are effective biosensor to spectrally differentiate multiple compounds by Surface-enhanced
Raman scattering (SERS). Metal film over nanosphere (MFON) has been demonstrated to exhibit reproducible and
predictable Raman enhancement. MFON can be fabricated using an economical process in which polystyrene (PS)
nanospheres are self-assembled on a planar solid supports and then followed by metal coating. In this work, we
investigate the MFON substrates with bimetallic coating to combine the optical-enhancing and stability features from Ag
and Au layers. The SERS responses are then quantified from the resultant bimetallic structures with 2-Naphthalenethiol.
We show that the bimetallic substrate of optimal Au/Ag thickness ratio renders SERS enhancement and stability
exceeding those of the Au-coated MFON. Compared to Au array, the bimetallic substrate exhibits quasi-bimetallic
nanoparticles of surpassing SERS (2.5 times) with enhancement factor determined to be 2×107. As a proof-of-concept for
biosensing in microfluidics, SERS nanotag was prepared and tested on the optimized BMFON. In addition, we propose a
fabrication scheme to construct MFON with alternating sizes (100nm and 400nm) of nanosphere. At optimal
proportional amount, the 100nm-spheres were packed within the gaps between the 400nm-spheres. The resultant
morphology renders additional nanogaps that could possibly lead to increment in SERS enhancement.
Temporal changes in signal intensity of Surface Enhanced Raman Scattering (SERS) upon laser excitation is an
interesting phenomenon in plasmonics. In-depth understanding of the phenomena is highly important especially when
developing a SERS sensor based on the intensity variation of particular Raman peak/band. One of the main challenges in
such a technique is the intensity reduction at a given location upon consecutive measurements. Previously, signal loss in
SERS measurement was attributed to the electric-field induced roughness relaxations in the SERS active surface. In such
cases, as the surface is smoothened out, signals are completely lost. In our observation, the reduction in the spectral
intensity is irreversible but never completely lost and a major part of it can be attributed to the plasmon induced heating
effect. Here, we experimentally demonstrate this effect by studying the SERS signal from four different Raman active
molecules adsorbed onto substrates that contain uniform nano-roughened bi-metallic silver/gold coating. Possible
mechanism that leads to irreversible signal loss is explained. Moreover, solutions for minimising such plasmonic heating
when developing a biosensor are also discussed.
A successful detection of inherently weak Raman signal from molecules is possible with giant enhancement of signal by the process of surface-enhanced Raman scattering (SERS). The SERS-induced enhancement is typically achieved when the molecules adsorbed onto the surface of a noble-metal substrate with nanometric roughness. Such SERS-substrate could be economically fabricated by convective assembly of polystyrene beads followed by metal deposition. The
characterization of mono-metallic substrate showed that the SERS enhancement factor increases with increasing
thickness of Ag or Au, with Ag-substrate giving the greatest SERS enhancement. However, the formation of silver oxide
layer could reduce the shelf-life of the Ag-substrate. Alternatively, Au is also used as the coating material owing to its
chemical inertness and biocompatibility. Despite the decent enhancement of the Au-substrate, Au-layer was found to be
unstable after prolonged incubation in crystal violet solution. The inherent deficiency in adhesiveness of Au to the glass
limits its use as a reliable and cost-effective substrate. In an attempt to improve the SERS-substrate, bimetallic substrate
was fabricated by depositing the Au-film, as a protective layer, on the Ag-substrate. In this case, the top layer of Au of
the bimetallic substrate remained intact after chemical treatment. Furthermore, the bimetallic substrate was shown to
give comparable level of enhancement as an Ag-substrate by choosing a proper thickness ratio of the bimetallic layers.
The result suggests that the design of bimetallic substrate could be optimized to maximize the SERS enhancement while
retaining a decent stability after laser illumination and chemical treatment. Our findings suggest that bimetallic substrates
are potentially useful for a reliable SERS-based biosensing.
The use of microfluidics for biofluid analysis offers a cheaper alternative to conventional techniques in disease diagnosis. However, traditional microfluidics design may be complicated by the need to incorporate separation elements into the system in order to facilitate specific molecular detection. Alternatively, an optical technique known as surface-enhanced Raman spectroscopy (SERS) may be used to enable identification of analyte molecules directly from a complex sample. This will not only simplify design but also reduce overall cost. The concept of SERS-based microfluidics is however not new and has been demonstrated previously by mixing SERS-active metal nanoparticles with a model sample, in situ, within the microchannel. Although the SERS reproducibility of these systems was shown to be acceptable, it is, however, not stable toward variations in the salt content of the sample, as will be shown in this study. We have proposed a microfluidics design whereby periodic SERS-active metal nanostructures are fabricated directly into the microchannel via a simple method of spin coating. Using artificial as well as human urine samples, we show that the current microfluidics is more stable toward variations in the sample's ionic strength.
Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve® enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.
Surface enhanced Raman spectroscopy (SERS) discovered some 30 years ago has gained popularity as a powerful analytical tool for developing chemo- and bio-sensing. The combination of SERS with the microfluidics technology can provide a miniaturized and portable device for bio-fluid analysis. However, as will be pointed out in this study, heat generated in a SERS-active substrate as a result of laser-induced plasmon resonance can unfavorably affect the sensitivity of a SERS-based microfluidic device. We will show that the plasmon-induced heat associated with SERS can significantly reduce the signal strength from the analyte under certain circumstances, and show heat-induced morphological changes in the SERS-active substrate as a primary cause of the observed signal changes. This study indicates that sufficient heat dissipation is crucial for the proper working of a SERS-based microfluidic device.
Confocal endomicroscopy is a novel, noninvasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. This study was to explore the possibility of utilizing a novel rigid confocal endomicroscope (RCE) system for detecting morphological changes in living normal and neoplastic human and murine tongue tissue in combination with different photosensitizers, i.e. hypericin and 5-aminolevulinic acid (ALA) induced endogenous protoporphyrin IX (PPIX) fluorescence. Subjects were topically or systemically applied photosensitizer to the oral mucosa, and then fluorescence confocal endomicroscopy was performed on the tongue using the RCE system with the laser excitation wavelength at 488 nm. The preliminary results showed that confocal fluorescence images of the tongue can be acquired in real-time with well-defined micro-morphological structures, and changes of tissue structures associated with cancer transformation can also be identified. This study suggests that photosensitizer-mediated confocal endomicroscopy have a significant potential for rapid, non-invasive detection of early oral cancers in vivo.
Worldwide, oral cancer is the sixth most common cancer for both sexes. In Singapore, the 5-year survival rate of oral cancer is about 50%. The high mortality rate has been attributed to the difficulties in detecting the disease in an early treatable stage. Currently, the standard screening procedures for oral cancer are histopathology examination of biopsied tissues and exfoliative cytological assessment. These techniques, unfortunately, are low in sensitivity. In this study, we exploit the high amplification factor of SERS to investigate on the possibility of utilising molecular vibrational information from saliva samples to detect oral cancer early. All raw saliva samples were centrifuged at 13,000 krpm for 5 minutes to remove unwanted particles prior to SERS measurements. The purified saliva samples were then applied directly on gold particle films, followed by excitation with a 633 nm HeNe laser. SERS spectrum can be obtained in less than 2 minutes for each sample. We have studied the saliva spectra acquired from 5 normal individuals and 5 patients with oral cancer. In addition, we also observe new peaks at 1097 cm-1 and 1627 cm-1 in some of the abnormal samples. These peaks are not present in the spectra acquired from the normal samples. Preliminary measurements will be presented. This study may lead to the development of a sensitive and portable diagnostics system for oral cancer.
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