Malini Olivo

Ultrasensitive Near‐Infrared Raman Reporters for SERS‐Based In Vivo Cancer Detection

Surface-enhanced Raman spectroscopy (SERS) has recently emerged as an alternative to fluorescence-based spectroscopy in bioimaging, as it can minimize photobleaching, peak overlapping, and low signal-to-noise ratio in complex biological systems. SERS probes are based on the 10–10-fold scattering enhancement caused by the proximity of Ramanactive signature molecules to the surface of metal nanoparticles (NPs), which can be modulated with molecular recognition motifs to render diagnostic tools for optical imaging and therapeutic studies. However, the preparation of ultrasensitive SERS probes is hampered by the limited availability, sensitivity, and reproducibility of Raman-active compounds. This drawback is particularly important at the near-infrared (NIR) region, where the availability of reporters is restricted to a few Raman-active molecules. Herein, we report the first combinatorial approach to discover novel and highly sensitive NIR SERS reporters. The synthesis and screening of an 80-member tricarbocyanine library led to the identification of CyNAMLA-381 as a NIR SERS reporter with 12-fold higher sensitivity than the standard 3,3’-diethylthiatricarbocyanine (DTTC), and we validated its advantages for the construction of ultrasensitive in vivo SERS probes. A major bottleneck in SERS probe discovery is the development of highly sensitive Raman reporters. Most of the commonly used Raman signature molecules are active in the UV/Vis range (e.g., crystal violet, malachite green isothiocyanate, rhodamine-6G, Nile blue, 2-napthalenethiol, TRITC (tetramethylrhodamine-5-isothiocyanate), and XRITC (Xrhodamine-5-(and-6)-isothiocyanate), and thus have a restricted potential for in vivo imaging. The adequacy of the NIR region for in vivo studies has raised the interest in NIR surface-enhanced resonance Raman spectroscopy (SERRS)-active molecules. Although the cyanine derivative DTTC has been regarded as a standard in NIR SERRS studies, it shows only a moderate Raman intensity, which limits the preparation of highly sensitive probes for in vivo applications. Since little is known about the correlation between the cyanine scaffold and its Raman intensity, we designed a library of structurally diverse tricarbocyanines with the aim of discovering novel NIR SERRS-active compounds that surpass the sensitivity of DTTC. The tricarbocyanine core is an accessible NIR structure, the central chlorine atom of which can be replaced with different nucleophiles. We designed the synthesis of tricarbocyanine derivatives by substitution with different amines, and acetylated the resulting alkylor benzylamino groups to obtain compounds with NIR absorption properties and good chemical stability in aqueous media (CyNA). To prepare compounds that could be chemisorbed on gold nanoparticles (AuNPs), we prepared the scaffold 1 with an aminopropyl linker that could be later coupled to a disulfide-containing lipoic acid spacer (Scheme 1). The amine group of 1 was Boc-protected prior to the derivatization of the central chlorine atomwith 80 structurally different primary amines including heterocyclic, alkyl, and aromatic groups (for structures, see Chart S1 in the Supporting Information). After acetylation, the compounds were treated with an optimized TFA/dichloromethane (1:9) solution that overcame the lability of the tricarbocyanine core in acidic conditions. The final coupling to a lipoic acidactivated ester resin yielded 80 derivatives (CyNAMLA) with an average purity of 90% (for data of HPLC-determined purities, see Table S1 in the Supporting Information). CyNAMLA compounds proved to be remarkably NIRactive with absorbance maximumwavelengths around 800 nm (Table S1 in the Supporting Information). Their SERS [*] A. Samanta, X. Liao, Prof. Y. T. Chang Department of Chemistry & MedChem Program of Life Sciences Institute, National University of Singapore 117543 Singapore (Singapore) Fax: (+65)6779-1691 E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

Surface Plasmon Resonance Imaging Sensors: A Review

Surface plasmon resonance (SPR) imaging sensors realize label-free, real-time, highly sensitive, quantitative, high-throughput biological interaction monitoring and the binding profiles from multi-analytes further provide the binding kinetic parameters between different biomolecules. In the past two decades, SPR imaging sensors found rapid increasing applications in fundamental biological studies, medical diagnostics, drug discovery, food safety, precision measurement, and environmental monitoring. In this paper, we review the recent advances of SPR imaging sensor technology towards high-throughput multi-analyte screening. Finally, we describe our multiplex spectral-phase SPR imaging biosensor for high-throughput biosensing applications.

Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters.

SERS nanotags have been prepared to accomplish the multiplex detection of cancer cells. Herein we evaluated the adequacy of lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA) to function as multiplex partners with a triphenylmethine Raman reporter (B2LA) under a single excitation wavelength. SERS experiments enabled the multiplex recognition of two different cancer cells with antibody-conjugated nanotags that were derivatized with optimized cyanine and triphenylmethine reporters.

Sensitive SERS glucose sensing in biological media using alkyne functionalized boronic acid on planar substrates

In this work, we propose a novel glucose binding mechanism on a highly sensitive SERS substrate, in order to overcome challenges in specific glucose detection in bio-fluids. We make use of phenylboronic acid as a receptor for saccharide capture onto the substrate and the ability of the captured glucose molecule to undergo secondary binding with an alkyne-functionalized boronic acid to form a glucose-alkyne-boronic acid complex. The formation of this complex shows high selectivity for glucose, over other saccharides. In addition, the alkyne group of the alkyne-functionalized boronic acid exhibits a distinct Raman peak at 1996 cm(-1) in a biological silent region (1800-2800 cm(-1)) where most endogenous molecules, including glucose, show no Raman scattering, thus offering a high sensitivity over other SERS glucose sensing. The substrate offers long-term stability, as well as high SERS enhancement to the glucose-alkyne boronic acid complex on substrate. In addition, the reversibility of SERS signals at various incubation stages also shows reusability capabilities, whereas positive results in clinical urine samples demonstrate clinical feasibility. All these strongly suggest that this newly developed SERS-based assay offers great potential in glucose sensing.

Highly sensitive SERS detection and quantification of sialic acid on single cell using photonic-crystal fiber with gold nanoparticles

An ultrasensitive surface enhanced Raman spectroscopy (SERS) based sensing platform was developed to detect the mean sialic acid level on the surface of single cell with sensitivity as low as 2 fmol. This platform adopted the use of an interference-free Raman tag, 4-(dihydroxyborophenyl) acetylene (DBA), which selectively binds to sialic acid on the cell membrane. By loading the side channel of a photonic crystal fiber with a mixture of gold nanoparticles and DBA-tagged HeLa cell, and subsequently propagating laser light through the central solid core, strong SERS signal was obtained. This SERS technique achieved accurate detection and quantification of concentration of sialic acid on a single cell, surpassing previously reported methods that required more than 10(5) cells. Moreover, this platform can be developed into a clinical diagnostic tool to potentially analyze sialic acid-related diseases such as tumor malignancy and metastasis in real-time.

Photolon™ --photosensitization induces apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes.

The localization of photosensitizers in the subcellular compartments during photodynamic therapy (PDT) plays a major role in the cell destruction; therefore, the aim of this study was to investigate the intracellular localization of Chlorin e6-PVP (Photolon™) in malignant and normal cells. Our study involves the characterization of the structural determinants of subcellular localization of Photolon, and how subcellular localization affects the selective toxicity of Photolon towards tumor cells. Using confocal laser scanning microscopy (CLSM) and fluorescent organelle probes; we examined the subcellular localization of Photolon™ in the murine colon carcinoma CT-26 and normal fibroblast (NHLC) cells. Our results demonstrated that after 30xa0min of incubation, the distribution of Photolon was localized mainly in the cytoplasmic organelles including the mitochondria, lysosomes, Golgi apparatus, around the nuclear envelope and also in the nucleus but not in the endo-plasmic reticulum whereas in NHLC cells, Photolon was found to be localized minimally only in the nucleus not in other organelles studied. The relationship between subcellular localization of Photolon and PDT-induced apoptosis was investigated. Apoptotic cell death was judged by the formation of known apoptotic hallmarks including, the phosphatidylserine externalization (PS), PARP cleavage, a substrate for caspase-3 and the formation of apoptotic nuclei. At the irradiation dose of 1 J/cm2, the percentage of apoptotic cells was 80%, respectively. This study provided substantial evidence that Photolon preferentially localized in the subcellular organelles in the following order: nucleus, mitochondria, lysosomes and the Golgi apparatus and subsequent photodamage of the mitochondria and lyso-somes played an important role in PDT-mediated apoptosis CT-26 cells. Our results based on the cytoplasmic organelles and the intranuclear localization extensively enhance the efficacy of PDT with appropriate photosensitizer and light dose and support the idea that PDT can contribute to elimination of malignant cells by inducing apoptosis, which is of physiological significance.

Non-labeling multiplex surface enhanced Raman scattering (SERS) detection of volatile organic compounds (VOCs)

In this paper, we report multiplex SERS based VOCs detection with a leaning nano-pillar substrate. The VOCs analyte molecules adsorbed at the tips of the nano-pillars produced SERS signal due to the field enhancement occurring at the localized surface plasmon hot spots between adjacent leaning nano-pillars. In this experiment, detections of acetone and ethanol vapor at different concentrations were demonstrated. The detection limits were found to be 0.0017 ng and 0.0037 ng for ethanol and acetone vapor molecules respectively. Our approach is a non-labeling method such that it does not require the incorporation of any chemical sensing layer for the enrichment of gas molecules on sensor surface. The leaning nano-pillar substrate also showed highly reproducible SERS signal in cyclic VOCs detection, which can reduce the detection cost in practical applications. Further, multiplex SERS detection on different combination of acetone and ethanol vapor was also successfully demonstrated. The vibrational fingerprints of molecular structures provide specific Raman peaks for different VOCs contents. To the best of our knowledge, this is the first multiplex VOCs detection using SERS. We believe that this work may lead to a portable device for multiplex, specific and highly sensitive detection of complex VOCs samples that can find potential applications in exhaled breath analysis, hazardous gas analysis, homeland security and environmental monitoring.

A rapid and label-free SERS detection method for biomarkers in clinical biofluids.

A metal carbonyl-functionalized nanostructured substrate can be used in a rapid and simple assay for the detection of A1AT, a potential biomarker for bladder cancer, in clinical urine samples. The assay involves monitoring changes in the carbonyl stretching vibrations of the metal carbonyl via surface-enhanced Raman spectroscopy (SERS). These vibrations lie in the absorption spectral window of 1800-2200 cm(-1), which is free of any spectral interference from biomolecules.

Organometallic carbonyl clusters: a new class of contrast agents for photoacoustic cerebral vascular imaging

We report, for the first time, the use of a water-soluble organometallic compound as a stable, reliable and high-contrast photoacoustic contrast agent. This gives a significantly higher contrast than that achievable with alternatives such as single-walled carbon nanotubes. Image enhancement of the rat cerebral cortex vasculature was observed using in vivo dark-field photoacoustic microscopy.

Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma.

Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.