Kiang Wei Kho

Clinical SERS: are we there yet?

Surface Enhanced Raman Spectroscopy or SERS has witnessed many successes over the past 3 decades, owing particularly to its simplicity of use as well as its highly-multiplexing capability. This article provides an overview of SERS and its applicability in the field of bio-medicine. We will preview recent developments in SERS substrate designs, and the various sensing technologies that are based on the SERS phenomenon. An overview of the clinical applications of SERS is also included. Finally, we provide an opinion on the future trends of this unique spectroscopic technique.

Polymer-based microfluidics with surface-enhanced Raman-spectroscopy-active periodic metal nanostructures for biofluid analysis

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 samples ionic strength.

Chlorin e6-polyvinylpyrrolidone mediated photodynamic therapy—A potential bladder sparing option for high risk non-muscle invasive bladder cancer

BACKGROUND Bladder sparing treatment options for high risk non-muscle invasive blader cancer (NMIBC) after intravesical Bacillus Calmette-Guerin (BCG) failure are limited. OBJECTIVE To evaluate photodynamic therapy (PDT) using chlorin e6-polyvinylpyrrolidone (Ce6-PVP) as a bladder sparing therapy for NMIBC refractory to intravesical BCG therapy. MATERIALS AND METHODS Between July 2004 and June 2009, patients with recurrent NMIBC after induction intravesical BCG therapy were treated with PDT performed with a 665nm laser and light dosimetry of 10-24J/cm(2). The patients underwent cystoscopic surveillance for tumour recurrence post PDT. Post treatment lower urinary tract symptoms and bladder capacity were also monitored. Serum and urine samples were collected for spectrometric quantification of photosensitizer levels. RESULTS Five patients underwent PDT, with a total of seven treatments performed. One patient received intravenous Ce6-PVP, while the rest received intravesical Ce6-PVP.The median age was 80 years (mean 79 years, range 72-88 years). There were three patients with primary CIS of the bladder and two with T1 high grade TCC and CIS of the bladder. At a median follow-up of 29 months (mean 25 months, range 6-36 months), two patients were disease free, two patients developed recurrence and one patient progressed to muscle invasive disease. There were no immediate adverse effects. The patient receiving intravenous Ce6-PVP developed an enterovesical fistula 16 months post PDT. CONCLUSIONS Despite being a small pilot study, intravesical Ce6-PVP mediated PDT is a feasible bladder sparing treatment option for recurrent high risk non-muscle invasive bladder carcinoma in selected individuals.

APPLICATIONS OF GOLD NANOPARTICLES IN THE EARLY DETECTION OF CANCER

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. Here, we present two application examples of gold nanoparticles in the early detection of oral cancer. In the first, gold nanoparticles were used as a reflective contrast agent for performing molecular imaging under confocal reflectance microscopy for the early diagnosis of epithelial carcinoma. While in the second, closely-packed gold nanoparticle, films were used as a bio-sensing surface for the chemical analysis of saliva via Surface Enhanced Raman Scattering. Preliminary results will be discussed.

Surface enhanced Raman spectroscopic (SERS) study of saliva in the early detection of oral cancer

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.

The effect of design parameters of metallic substrate on the reproducibility of SERS measurement for biosensing

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.

Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

Sub-micron free-standing metal slabs with dielectric nano-voids of arbitrary shapes embedded beneath atomically-flat surface

Thin metal slabs with plasmonic nano-voids buried within the skin depth (< 25 nm) of surface plasmon polaritons have been of theoretical as well as technical interests for many years due to its unique optical properties such as sharp absorbance dips and anti-crossing plasmonic dispersion characteristics. Unfortunately, such interesting plasmonic properties have not been experimentally reproduced, especially in the UV-Vis regime, owing to the involuntary surface roughness occurred in systems fabricated using conventional techniques. Here, we describe a versatile cryogenic-stripping approach for encapsulating a monolayer of nano-voids of virtually any arbitrary shapes underneath an atomically-smooth (δ < 0.55 nm) surface of a free-standing metal slab. By artificially varying the topography of the capping metal surface from ultra-smooth to moderately-rough, we show structural symmetricity in a nano-void-metal system can render the overall plasmonic responses becoming profoundly influenced by the surface smoothness. The current fabrication technique is thus of primary importance to the preparation of any kind of smooth nano-void-passivated metal slabs.

Hyper-spectral confocal nano-imaging with a 2D super-lens

Achieving sub-100 nm resolution over a broad visible bandwidth has long been an elusive goal in the nano-imaging of cell-surface interfaces. While metamaterial super-lenses and near-field optics have been previously demonstrated, these techniques can operate only at one wavelength, and do not provide accesses to the cell-surface interfaces. Here, we investigate a broadband 2D lens comprised of an oblate spheroidal dielectric cavity embedded just beneath a planar metal surface. The lens operates by adiabatically focusing asymmetric plasmon energies at sub-100 nm scale on the low-index side of the thin metal film formed between the cavity top and the planar metal surface. We then proposed the use of our lens in a high-resolution far-field confocal microscopy setup. Due to the surface-field nature of our lens, the presented system holds potential as an indispensable tool for cell-surface interfacial studies that require sub-100 nm hyper-spectral imaging analysis.

Fluctuation in surface enhanced Raman scattering intensity due to plasmon related heating effect

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.