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.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Facile synthesis of Raman active phospholipid gold nanoparticles

Gold nanoparticle-based surface-enhanced Raman scattering (SERS) probes have shown promise for disease detection and diagnosis. To improve their structural and functional stability for in vivo applications, we synthesized a colloidal SERS gold nanoparticle that encapsulates Raman molecules adsorbed on 60 nm gold with a nonthiol phospholipid coating. Transmission electron microscopy and Raman and UV spectroscopy validated its reproducibility and stability. This novel lipid-based SERS probe provides a viable alternative to the PEGylation and silica coating strategies.     

Tunable Assembly of Peptide-coated Gold Nanoparticles

The interaction between peptides and gold surfaces has increasingly been of interest for bionanotechnology applications. To more fully understand how to control such interactions, we have studied the optical properties of peptide-modified gold nanoparticles as a function of peptide composition, pH of the surrounding medium, and peptide concentration. We show using localized surface plasmon resonance, transmission electron microscopy, and surface-enhanced Raman scattering (SERS) that selected “gold-binding peptides” (GBPs), similar to those isolated for binding to gold films using yeast display, can bind to gold nanoparticles at a variety of pHs. Peptide modifications of nanoparticles can lead to irreversible particle aggregation when the pH of the solution is kept below the isoelectric point (pI) of the peptide. However, at pHs above the peptide’s pI, particles remain stable in solution, and peptides remain bound to the particles possibly through amine coordination of gold. Additionally, we demonstrate the potential in using SERS for the direct detection of GBPs on gold-silica nanoshells, eliminating the need for indirect labeling methods.     

SERS Properties of Different Sized and Shaped Gold Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa Extract

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.     

Label free sub-picomole level DNA detection with Ag nanoparticle decorated Au nanotip arrays as surface enhanced Raman spectroscopy platform

Label free optical sensing of adenine and thymine oligonucleotides has been achieved at the sub-picomole level using self assembled silver nanoparticles (AgNPs) decorated gold nanotip (AuNT) arrays. The platform consisting of the AuNTs not only aids in efficient bio-immobilization, but also packs AgNPs in a three dimensional high surface area workspace, assisting in surface enhanced Raman scattering (SERS). The use of sub-10 nm AgNPs with optimum inter-particle distance ensures amplification of the chemically specific Raman signals of the adsorbed adenine, thymine, cytosine and guanine molecules in SERS experiments. High temporal stability of the Raman signals ensured reliable and repeatable DNA detection even after three weeks of ambient desk-top conservation. This facile architecture, being three dimensional and non-lithographic, differs from conventional SERS platforms.     

Direct Fabrication Route to Plastic-Supported Gold Nanoparticles for Flexible NIR-SERS

Stable gold nanoparticles with surface plasmon resonance tunable from visible (Vis) to near-infrared (NIR) are deposited via a direct sputtering methodology on large area polyethylene terephthalate (PET) to be used as effective, flexible NIR surface-enhanced Raman scattering (SERS) substrates. An O₂ plasma treatment of PET is used to tailor growth dynamics, geometry, and plasmonic properties of nanoparticles. The O₂ plasma treatment of PET results also in effective improvement of nanoparticle anchoring on the plastic substrate, providing more stable, flexible SERS systems. The functionality of fabricated SERS substrates has been tested using benzylthiol, and SERS enhancement factors in the range 10⁴ have been achieved, which are comparable with those reported in literature for gold nanostructures fabricated on silicon substrate. These results attest the great potentiality of this methodology for the production of cost-effective flexible and reusable large-scale SERS substrates.     

Functionalized Plasmonic Anisotropic Nanocrystals for Multimodal Imaging of Cancer Cells

Nanoparticles internalized by cells are valuable probes for bioimaging. In particular, nanoparticles can be detected in “biological transmission window,” i.e., near infrared region. Here, we report a preparation of biotargeting diethylthiatricarbocyanine iodide (DTTC)-functionalized gold nanorods, utilized for detection of malignant cells. These biotargeting DTTC-functionalized gold nanorods are efficiently internalized into cultured cells and can serve as probes for surface-enhanced Raman scattering (SERS) and dark-field imaging. The robust SERS signal from malignant cells has clearly demonstrated a signature peak of DTTC in the presence of our formulation. A short acquisition time, we used in this experiment, is able to exclude bulk of Raman signal from natural cellular constituents. This signature peak will be a key of identifying cancer due to cancer-specific property of biotargeted molecule. The results are leading to promising real-time cancer detection. In addition, these multimodal probes demonstrated low toxicity in cell viability studies which enables a broad range of multiplex imaging applications.     

表面增强拉曼光谱技术在肿瘤病理中的应用

表面增强拉曼散射（Surface-enhanced Raman Scattering,SERS）技术作为鉴定生物分子种类最有力的分析工具之一,具有灵敏度高、特异性强、稳定性好及检测条件温和等优点。目前,SERS技术在肿瘤病理领域的应用尚处于起步阶段,但已显现出良好的应用前景和发展空间。该文简要介绍了SERS的机理、特性及活性基底,并对SERS技术在肿瘤病理的研究进展、局限性及潜在应用价值方面做较为全面的综述。     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

表面增强拉曼光谱在DNA检测中的应用

随着光学技术的发展,表面增强拉曼光谱(SERS)作为一种新兴的技术被逐渐应用于生物医学领域。SERS波谱作为一种振动波谱,能够反应被测物质的内部信息,具有指纹识别特征;具备高灵敏度、高效能的特点,且能实现复合样本的同时测定;带标记的SERS技术能进一步提高SERS检测的特异性。目前SERS技术已被广泛用于体内外DNA、蛋白分子的检测,为生物分子的分析检测提供了一种崭新、高效的手段。     

Characterization and application of Raman labels for confocal Raman microspectroscopic detection of cellular proteins in single cells

A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.     

A Shape-Engineered Surface-Enhanced Raman Scattering Optical Fiber Sensor Working from the Visible to the Near-Infrared

Surface-enhanced Raman scattering (SERS) takes advantage of the giant electromagnetic field enhancement provided by localized surface plasmons in metal nanoparticles to amplify the weak Raman scattering of the molecules. Optical fibers coated with noble metal nanoparticles can therefore be used as SERS-based sensors for remote detection of molecular species. In this article, we report on the development of an optical fiber SERS sensor capable to operate on a range of excitation wavelengths from the visible to the near-infrared. We introduce a quasistatic chemical etching protocol to engineer the tip shape and investigate the effects of the tip shape on the sensor performances.     

Dual-mode probe based on mesoporous silica coated gold nanorods for targeting cancer cells

A dual-mode imaging probe for targeting cancer cells has been fabricated based on mesoporous silica coated gold nanorods (MS-GNRs) for the first time. In this probe, fluorescence and surface enhanced Raman scattering (SERS) signals can be generated independently by using different excitation wavelengths. To investigate the targeting performance of the probe, folic acid (FA) is conjugated on the outer surfaces of MS-GNRs as a targeting ligand and HeLa cells were used as model cancer cells because they overexpress folate receptors (FRs). The endocytosis mechanism was verified by competing experiments with free FA through both fluorescence images and SERS mappings. Moreover, the cytotoxicity of the probe was remarkably reduced in comparison with the GNRs without the silica shell as proved by the results of MTT assay. Compared with traditional imaging probes, this new type of nanoprobe has great potential for multiplexed imaging in living cells, which can be easily realized by using fluorescence and SERS signals.     

Surface enhanced Raman scattering for narcotic detection and applications to chemical biology

Raman spectroscopy is rapidly finding favour for applications in the life science because of the ease with which it can be used to extract significant data from tissue and cells. However, the Raman effect is an inherently weak effect, which hinders the analysis of low concentration analytes. Raman sensitivity can be improved via the surface enhanced Raman scattering (SERS) effect. In SERS, Raman spectra are dramatically amplified when a molecule is adsorbed onto nano-roughened noble metal surfaces such as silver and gold. The degree of enhancement enables single-molecule detection, which offers the potential for the unambiguous identification of analytes at concentrations that are useful in both a forensic and a chemical biology context. Here we discuss some of the practical applications of SERS to both low-level narcotic detection, and how this can be applied to chemical biology.     

Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay

DNA methylation is a key diagnostic maker for genetic disease, cancer progression and pharmcogenomics. So far various techniques have been developed for DNA methylation assay, but most of them are laborious and time-consuming. Here we develop a simple and highly sensitive DNA methylation assay based on single base extension reaction and surface enhanced Raman spectroscopy (SERS). In the presence of methylated DNA, gold nanoparticle-modified capture probe can couple with a cyanine 5-deoxyribonucleoside triphosphate (cy5-dGTP) through single base extension reaction, and generates a high SERS signal after further addition of gold nanoparticles to increase the local electromagnetic field. While in the presence of unmethylated DNA, gold nanoparticle-modified capture probe cannot couple with cy5-dGTP due to the presence of a mismatch base, and no SERS signal is observed. This single base extension reaction-based SERS can determine methylated DNA with a detection limit of 3 pM, and can even distinguish as low as 1% methylation level in tumor suppressor gene CDKN2/p16/MTS1 (p16) from the mixtures. Notably, the sensitivity of this assay has improved by 5 orders of magnitude as compared to reported gold nanoparticle-based colorimetric assay, and by 2 orders of magnitude as compared to microarray-based methylation-sensitive single nucleotide primer extension assay (Ms-SNuPE). This method might be further applied to detect the methylation status in tumor-linked genes for cancer diagnosis.     

A new protein A assay based on Raman reporter labeled immunogold nanoparticles

A unique, sensitive, highly specific, and photobleaching-resistant immunoassay system utilizing gold nanoparticles and surface-enhanced Raman scattering (SERS) is described. This new system, featuring a capability of bifunctional analysis, is manufactured by chemisorption of antibody immunoglobulin G (IgG) on gold nanoparticles (AuNP), followed by coupling the Raman-active reporter molecule, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the surface of IgG-AuNP. The adsorbed DTNB molecules exhibit strong Raman signals via both electromagnetic and chemical enhancement. The narrow spectral widths and high photostability assure the system to be an excellent detection label. This SERS-based immunoassay is applied to the detection of protein A, which is a specific surface antigen of Staphylococcus aureus. A working curve is obtained by plotting the intensity of the SERS signal of symmetric NO(2) stretching of DTNB at 1333cm(-1) versus the concentration of the analyte (antigen). A dynamic range of two to three orders of magnitude and a detection limit of 1pg/mL of protein A are achieved.     

Interface-Induced Ag Monolayer Film for Surface-Enhanced Raman Scattering Detection of Water-Insoluble Enrofloxacin

Water-insoluble molecules usually show poor surface-enhanced Raman scattering (SERS) signals, because they are hardly adsorbed on the surface of most commonly used SERS substrates, such as aqueous Ag or Au colloids. In this work, a highly sensitive and reproducible Ag monolayer film (Ag MLF) SERS substrate prepared by self-assembly of Ag nanoparticles (Ag NPs) on water/oil interface can realize the trace SERS detection of water-insoluble enrofloxacin. The positively charged phase transfer catalyst can transfer the negatively charged Ag nanoparticles in aqueous solution to the water/oil interface. At the same time, the water-insoluble enrofloxacin can also be attracted to the interface because of its lipophilic group. The type/volume of the oil phase and phase transfer catalyst and the vortex mixing time were all optimized to maximize the SERS effect of Ag MLF. Results showed that trace water-insoluble enrofloxacin can be identified by Ag MLF and its detection sensitivity was significantly improved. The proposed novel Ag MLF can be further applied to detect other water-insoluble molecules in SERS.

     

A Real-Time Clinical Endoscopic System for Intraluminal,Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.