U. S. Dinish

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

Actively Targeted In Vivo Multiplex Detection of Intrinsic Cancer Biomarkers Using Biocompatible SERS Nanotags

Surface-enhanced Raman scattering (SERS) technique is becoming highly popular for multiplex biosensing due to the ‘fingerprint’ Raman spectra from every molecule. As a proof-of-concept, we demonstrated the actively targeted multiplex in vitro and in vivo detection of three intrinsic cancer biomarkers - EGFR, CD44 and TGFβRII in a breast cancer model using three multiplexing capable, biocompatible SERS nanoparticles/nanotags. Intra-tumorally injected antibody conjugated nanotags specifically targeting the three biomarkers exhibited maximum signal at 6 hours and no detectable signal at 72 hours. However, nanotags without antibodies showed no detectable signal after 6 hours. This difference could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. Thus, this study establishes SERS nanotags as an ultrasensitive nanoprobe for the multiplex detection of biomarkers and opens up its potential application in monitoring tumor progression and therapy and development into a theranostic probe.

Development of highly reproducible nanogap SERS substrates: Comparative performance analysis and its application for glucose sensing

We report a new class of a SERS substrate with ordered nanostructures fabricated on silicon wafer using a deep UV (DUV) lithography technique followed by surface coating of silver and/or gold film. These substrates possess sharp edged nanogaps, which are responsible for the SERS enhancement. SERS performance of these substrates was analyzed by studying its reproducibility, repeatability and signal enhancement measured from 2-naphthalene thiol (NT) molecule covalently anchored on to the substrate. SERS performance of this substrate was also compared with a commercial substrate and metal film over nanosphere (MFON) substrate, which is one of the most promising reported substrates. It was found that MFON substrate showed a slightly higher SERS intensity among all three chosen substrates, but the relative standard deviation (RSD) of the intensity for the two prominent peaks of NT was about 7-14% while for our nanogap DUV substrate the RSD was less than 3% with comparable SERS signal intensities to MFON. For the commercial substrate, the relative standard deviation was about 7-9% but with a much lower SERS signal intensity. To our knowledge, this observed reproducibility along with good SERS enhancement with nanogap substrate is the best among the reported SERS substrates. These observed results with the nanogap substrate show great potential for the development of a sensitive SERS biosensing platform. Efficacy of the nanogap DUV substrate for biosensing was demonstrated for in vitro glucose sensing under physiologically relevant conditions.

Development of biocompatible SERS nanotag with increased stability by chemisorption of reporter molecule for in vivo cancer detection.

Biocompatible surface-enhanced Raman scattering (SERS) nanotag has been developed by chemisorption of novel Raman reporters on gold colloid. We modified our previously published best five reporter molecules (B2, B7, C3, C7 and C9) from triphenylmethine (TM) library using lipoic acid (LA) as a linker to covalently attach the reporters on gold colloid. Among these TM-LA molecules, B2LA showed the highest SERS signal intensity and stability over time. Further, time course SERS intensity of B2LA was compared with currently popular Raman reporter malachite green isothiocyanate (MGITC). The results demonstrated that signal intensity from B2LA was even stable over a period of one month. In vitro SERS screening was performed in cancer cell lines using B2LA containing nanotag conjugated with selective antibodies recognizing HER2 and EGFR cancer proteins. We found reasonably strong SERS signals from both HER2 and EGFR positive cells whereas no signal was measured from respective negative cells. Moreover, we successfully proved this recognition by cell imaging using fluorescein isothiocyanate (FITC) labeled antibody conjugated nanotag. Both SERS and cell-imaging study further confirmed the selective binding of antibody conjugated nanotag to cancer cells over-expressing HER2 and EGFR. In addition, as a proof of concept, in vivo SERS measurement in a mouse model was carried out to detect the nanotag-anchored cancer cells that are subcutaneously injected to the animal.

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber.

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ∼100 pg in a sample volume of ∼10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.

Multifunctional photosensitizer-based contrast agents for photoacoustic imaging.

Photoacoustic imaging is a novel hybrid imaging modality combining the high spatial resolution of optical imaging with the high penetration depth of ultrasound imaging. Here, for the first time, we evaluate the efficacy of various photosensitizers that are widely used as photodynamic therapeutic (PDT) agents as photoacoustic contrast agents. Photoacoustic imaging of photosensitizers exhibits advantages over fluorescence imaging, which is prone to photobleaching and autofluorescence interference. In this work, we examined the photoacoustic activity of 5 photosensitizers: zinc phthalocyanine, protoporphyrin IX, 2,4-bis [4-(N,N-dibenzylamino)-2,6-dihydroxyphenyl] squaraine, chlorin e6 and methylene blue in phantoms, among which zinc phthalocyanine showed the highest photoacoustic activity. Subsequently, we evaluated its tumor localization efficiency and biodistribution at multiple time points in a murine model using photoacoustic imaging. We observed that the probe localized at the tumor within 10 minutes post injection, reaching peak accumulation around 1 hour and was cleared within 24 hours, thus, demonstrating the potential of photosensitizers as photoacoustic imaging contrast agents in vivo. This means that the known advantages of photosensitizers such as preferential tumor uptake and PDT efficacy can be combined with photoacoustic imaging capabilities to achieve longitudinal monitoring of cancer progression and therapy in vivo.

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.

Design and fabrication of random silver films as substrate for SERS based nano-stress sensing of proteins

We report a simple and easy to fabricate random silver film (RSF) as a highly sensitive Surface Enhanced Raman Scattering (SERS) substrate which can be fabricated directly onto a dielectric substrate such as glass. An electron beam evaporation system was used for substrate fabrication. The SERS activity is attributed to the formation of electromagnetic ‘hot-spots’ on the film. Substrate performance is analyzed by studying the reproducibility and signal enhancement from the Raman active molecule, 2-naphthalene thiol (NT), which is covalently anchored to the substrate. The metal thickness is optimized to achieve the highest SERS enhancement. Based on this study we found that a 7 nm RSF substrate gave the best SERS activity. The SERS signal intensity exhibited by 7 nm RSF is found to be at least 3 orders of magnitude higher than that of a commercial substrate. The SERS enhancement factor is estimated to be ∼1 × 107 with a point-to-point intensity variation of about 12% and it reaches a maximum of 15% for batch-to-batch comparison. The efficacy of this substrate for biosensing is demonstrated by detecting H1 influenza protein, and the detection limit is found to be ∼10 pM when it is used along with a recently established nano-stress SERS sensor, 4-ATP (4-amino-thiophenol), as linker molecule. This detection limit shows a performance superior to conventional ELISA (which has a nM detection limit). These results show promise for the development of a biosensing platform based on the marriage of RSF with nano-stress sensors.

Molecular photoacoustic imaging of breast cancer using an actively targeted conjugated polymer

Conjugated polymers (CPs) are upcoming optical contrast agents in view of their unique optical properties and versatile synthetic chemistry. Biofunctionalization of these polymer-based nanoparticles enables molecular imaging of biological processes. In this work, we propose the concept of using a biofunctionalized CP for noninvasive photoacoustic (PA) molecular imaging of breast cancer. In particular, after verifying the PA activity of a CP nanoparticle (CP dots) in phantoms and the targeting efficacy of a folate-functionalized version of the same (folate-CP dots) in vitro, we systemically administered the probe into a folate receptor-positive (FR+ve) MCF-7 breast cancer xenograft model to demonstrate the possible application of folate-CP dots for imaging FR+ve breast cancers in comparison to CP dots with no folate moieties. We observed a strong PA signal at the tumor site of folate-CP dots-administered mice as early as 1 hour after administration as a result of the active targeting of the folate-CP dots to the FR+ve tumor cells but a weak PA signal at the tumor site of CP-dots-administered mice as a result of the passive accumulation of the probe by enhanced permeability and retention effect. We also observed that folate-CP dots produced ~4-fold enhancement in the PA signal in the tumor, when compared to CP dots. These observations demonstrate the great potential of this active-targeting CP to be used as a contrast agent for molecular PA diagnostic imaging in various biomedical applications.

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.