Gold-coated array patterned with tightly-packed nanospheres was developed as a substrate base for constructing SERSenriched
nanogaps with Au-nanoparticles (GNPs). Using 1,2-ethanedithiol as a linker, Au-NPs (=17-40nm) were
anchored covalently on the sphere-array. Thin Au layer was sputtered on the substrate to mask the citrate coating of
GNPs that could demote the sensing mechanism. The negatively-charged GNP surface warrants the colloidal stability,
but the resulting repulsive force keeps the immobilized NPs apart by about 40nm. The attained gap size is inadequately
narrow to sustain any intense enhancement owing to the near-field nature of SERS. Minimal amount of NaCl was then
added to slightly perturb the colloidal stability by reducing their surface charge. Notably, the interparticle-gap reduces at
increasing amount of salt, giving rise to increased packing density of GNPs. The SERS enhancement is also found to
exponentially increase at decreasing gap size. Nevertheless, the minimum gap achieved is limited to merely 7nm.
Excessive addition of salt would eventually induce complete aggregation of particles, forming clustered NPs on the
array. A simple sputtering-growth approach is therefore proposed to further minimize the interparticle gap by enlarging
the seeded NPs based on mild sputtering. The SEM images confirm that the gap below 7nm is achievable. With advent
of the colloidal chemistry, the combined salt-induced aggregation and sputtering-growth techniques can be applied to
engineer interparticle gap that is crucial to realize an ultrasensitive SERS biosensor. The proposed two-step preparation
can be potentially adopted to fabricate the SERS-enriched nanogaps on the microfluidics platform.
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.
Surface Enhanced Raman Scattering (SERS) technique is used as an indispensable and sensitive modality for bio-sensing
due to its ability to distinguish the analyte molecules based on their distinct 'fingerprint' spectra. One of the most
promising SERS substrates for biosensing was fabricated by coating noble metal film over orderly packed nanospheres.
However, the major challenge in developing such a sensor is to achieve reproducible SERS substrate. Here, we report a
new class of SERS substrate with ordered 3D nanostructures fabricated on silicon wafer by deep UV lithography
technique followed by bi-metallic coating of silver and gold. Compared to the substrate fabricated by conventional
nanosphere lithography, this approach allows better control of the nanostructures, which in turn gives uniform surface
roughness for the metal film to provide adequate SERS enhancement with high reproducibility. Significance of this
substrate for biomedical application was demonstrated by glucose sensing under physiologically relevant conditions.
Partitioning and localization of glucose molecules within the first few nanometers of active SERS substrate was achieved
by a self assembled monolayer (SAM) on the surface of substrate.
We present the implementation of a fluorescence lifetime imaging microscopy (FLIM) system for cellular
characterisation. FLIM system can be used as an investigative tool to identify minor biochemical changes in cellular
abnormalities. These subtle changes could possibly alter cellular fluorescence properties such as emission wavelength
and lifetime. In this study, the fluorescence lifetime of haematoxylin and eosin (H&E)-stained tissues were investigated
using a wide-field time-domain FLIM system. The transient response of epithelial fluorescence was investigated and the
lifetime extracted using a bi-exponential model. It was found that the fluorescence lifetimes of eosin can be correlated to
the tissue histology. The preliminary result suggests that tumor-associated molecules are retained in the tissues even after
tissue fixation and staining. The developed FLIM system was successfully applied to detect the histological changes in
the tissue samples. Optimization of system parameters is also discussed.
Bladder cancer is the fourth common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate can be reduced by early treatment following precancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimen, which is both invasive and time-consuming. In this study ex vivo urine fluorescence cytology is investigated to offer a timely and biopsy-free means for detecting bladder cancers. Sediments in patients' urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cells from the other cells based on the cellular size. Intensity histogram of each targeted cell was plotted and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cells was observed. Diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this paper. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder
cancer diagnosis.
Applications involving fluorescence detection in point-of-care systems are both interesting and challenging in nature. The applications usually require a simple, compact, robust, highly sensitive yet affordable system. As a result, the system needs to be efficient in fluorescence detection by using practical and easily fabricated, hence inexpensive sensors. In this paper, a fluorescence sensor using an in-fiber microchannel has been developed and tested successfully. A blue LED, multimode PMMA or silica fiber, mini-PMT and fluorescein in PBS pH 7.4 buffer solution were used as the excitation source, light guide, fluorescence detector and sample, respectively. Microfluidic channels of 100μm width and 1cm length were fabricated in the optical fibers using a direct write CO2 laser system. The channels in the fibers were examined using a SEM and an optical microscope. Experimental results show that the sensor is highly sensitive, being able to detect 0.1 μg/L of fluorescein in the PBS buffer solution, with good signal to noise ratio and the results are reproducible. The data obtained using silica fibers as sensors when compared with the results from PMMA fibers show that the silica fiber sensor has better sensitivity than the PMMA fiber sensor. This could be due to the fouling effect created by the frosty layer at the bottom of the microchannel made within the PMMA fiber. Our future work will integrate the fiber sensor into microfluidic chips for lab-on-a-chip applications.
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