Detection of SERS active labelled DNA based on surface affinity to silver nanoparticles

Developments in specific DNA detection assays have been shown to be increasingly beneficial for molecular diagnostics and biological research. Many approaches use optical spectroscopy as an assay detection method and, owing to the sensitivity and molecular specificity offered, surface enhanced Raman scattering (SERS) spectroscopy has become a competitively exploited technique. This study utilises SERS to demonstrate differences in affinity of dye labelled DNA through differences in electrostatic interactions with silver nanoparticles. Results show clear differences in the SERS intensity obtained from single stranded DNA, double stranded DNA and a free dye label and demonstrate surface attraction is driven through electrostatic charges on the nucleotides and not the SERS dye. It has been further demonstrated that, through optimisation of experimental conditions and careful consideration of sequence composition, a DNA detection method with increased sample discrimination at lower DNA concentrations can be achieved.     

Optimization of the preparation of glass-coated, dye-tagged metal nanoparticles as SERS substrates

Dye-tagged metal nanoparticles are of significant interest in SERS-based sensitive detection applications. Coating these particles in glass results in an inert spectral tag that can be used in applications such as flow cytometry with significant multiplexing potential. Maximizing the SERS signal obtainable from these particles requires care in partitioning available nanoparticle surface area (binding sites) between the SERS dyes and the functionalized silanes necessary for anchoring the glass coating. In this article, we use the metal-mediated fluorescence quenching of SERS dyes to measure surface areas occupied by both dyes and silanes and thus examine SERS intensities as a function of both dye and silane loading. Notably, we find that increased surface occupation by silane increases the aggregative power of added dye but that decreasing the silane coverage allows a greater surface concentration of dye. Both effects increase the SERS intensity, but obtaining the optimum SERS intensity will require balancing aggregation against surface dye concentration.     

Facile fabrication of large area of aggregated gold nanorods film for efficient surface-enhanced Raman scattering

An effective and facile method for fabrication of large area of aggregated gold nanorods (AuNRs) film was proposed by self-assembly of AuNRs at a toluene/water interface for the first time. It was found that large area of aggregated AuNRs film could be formed at the interface of toluene and water due to the interfacial tension between the two phases. The obtained large area of aggregated AuNRs film exhibits strong surface-enhanced Raman scattering (SERS) activity with 4-aminothiophenol (4-ATP) and 2-aminothiophenol (2-ATP) as the probe molecules based on the strong electromagnetic coupling effect between the very adjacent AuNRs. Enhancement factors (EF) were used to estimate the SERS activity of the aggregated AuNRs film, which is obtained to be 1.7x10(5) for 7a vibration of 4-ATP. SERS intensity is compared with AuNRs deposited directly on glass, indicating high SERS activity and reproducibility of the aggregated AuNRs film. In addition, SERS activity has also been successfully demonstrated for dye molecule (Rhodamin 6G (R6G)) and biological small molecule (adenine) on the aggregated AuNRs film, showing great potential of the aggregated AuNRs film as a convenient and powerful SERS substrate for biological tags and biological molecular detection.     

Biocompatible Triplex Ag@SiO2@mTiO2 Core–Shell Nanoparticles for Simultaneous Fluorescence‐SERS Bimodal Imaging and Drug Delivery

Herein, we report the synthesis of biocompatible triplex Ag@SiO₂@mTiO₂ core–shell nanoparticles (NPs) for simultaneous fluorescence‐surface‐enhanced Raman scattering (F‐SERS) bimodal imaging and drug delivery. Stable Raman signals were created by typical SERS tags that were composed of Ag NPs for optical enhancement, a reporter molecule of 4‐mercaptopyridine (4‐Mpy) for a spectroscopic signature, and a silica shell for protection. A further coating of mesoporous titania (mTiO₂) on the SERS tags offered high loading capacity for a fluorescence dye (flavin mononucleotide) and an anti‐cancer drug (doxorubicin (DOX)), thereby endowing the material with fluorescence‐imaging and therapeutic functions. The as‐prepared F‐SERS dots exhibited strong fluorescence when excited by light at 460 nm whilst a stable, characteristic 4‐Mpy SERS signal was detected when the excitation wavelength was changed to longer wavelength (632.8 nm), both in solution and after incorporation inside living cells. Their excellent biocompatibility was demonstrated by low cytotoxicity against MCF‐7 cells, even at a high concentration of 100 μg mL^?1. In vitro cell cytotoxicity confirmed that DOX‐loaded F‐SERS dots had a comparable or even greater therapeutic effect compared with the free drug, owing to the increased cell‐uptake, which was attributed to the possible endocytosis mechanism of the NPs. To the best of our knowledge, this is the first proof‐of‐concept investigation on a multifunctional nanomedicine that possessed a combined capacity for fast and multiplexed F‐SERS labeling as well as drug‐loading for cancer therapy.     

Semi-quantitative analysis of indigo by surface enhanced resonance Raman spectroscopy (SERRS) using silver colloids

In this paper we report for the first time semi-quantitative analysis of indigo using surface enhanced Raman spectroscopy (SERS) and surface enhance resonance Raman spectroscopy (SERRS). Indigo, a dye widely used today in the textile industry, has been used, historically, both as a dye and as a pigment; the latter in both paintings and in printed material. The molecule is uncharged and largely insoluble in most solvents. The application of SERS/SERRS to the semi-quantitative analysis of indigo has been examined using aggregated citrate-reduced silver colloids with appropriate modifications to experimental protocols to both obtain and maximise SERRS signal intensities. Good linear correlations are observed for the dependence of the intensities of the SERRS band at 1151 cm(-1) using laser exciting wavelengths of 514.5 nm (R=0.9985) and 632.8 nm (R=0.9963) on the indigo concentration over the range 10(-7)-10(-5) and 10(-8)-10(-5) mol dm(-3), respectively. Band intensities were normalised against an internal standard (silver sol band at 243 cm(-1)). Resonance Raman spectra (RRS) of aqueous solutions of indigo could not be collected because of its low solubility and the presence of strong fluorescence. It was, however, possible to obtain RS and RRS spectra of the solid at each laser excitation wavelength. The limits of detection (L.O.D.) of indigo by SERS and SERRS using 514.5 and 632.8 nm were 9 ppm at both exciting wavelengths. Signal enhancement by SERS and SERRS was highly pH dependent due to the formation of singly protonated and possibly doubly protonated forms of the molecule at acidic pH. The SERS and SERRS data provide evidence to suggest that an excess of monolayer coverage of the dye at the surface of silver colloids is observed at concentrations greater than 7.85x10(-6) mol dm(-3) for each exciting wavelength. The data reported herein also strongly suggest the presence of multiple species of the indigo molecule.     

Silica-coated gold nanostars for combined surface-enhanced Raman scattering (SERS) detection and singlet-oxygen generation: a potential nanoplatform for theranostics

This paper reports the synthesis and characterization of surface-enhanced Raman scattering (SERS) label-tagged gold nanostars, coated with a silica shell containing methylene blue photosensitizing drug for singlet-oxygen generation. To our knowledge, this is the first report of nanocomposites possessing a combined capability for SERS detection and singlet-oxygen generation for photodynamic therapy. The gold nanostars were tuned for maximal absorption in the near-infrared (NIR) spectral region and tagged with a NIR dye for surface-enhanced resonance Raman scattering (SERRS). Silica coating was used to encapsulate the photosensitizer methylene blue in a shell around the nanoparticles. Upon 785 nm excitation, SERS from the Raman dye is observed, while excitation at 633 nm shows fluorescence from methylene blue. Methylene-blue-encapsulated nanoparticles show a significant increase in singlet-oxygen generation as compared to nanoparticles synthesized without methylene blue. This increased singlet-oxygen generation shows a cytotoxic effect on BT549 breast cancer cells upon laser irradiation. The combination of SERS detection (diagnostic) and singlet-oxygen generation (therapeutic) into a single platform provides a potential theranostic agent.     

Aversatile β-cyclodextrin functionalized silver nanoparticle monolayer for capture of methyl orange from complex wastewater

The toxic organic dye contaminants in wastewater are extremely harmful to the ecosystem.Surface enhanced Raman scattering(SERS) is a technique with high sensitivity and chemical specificity which fulfills the requirements for monitoring dye contaminants in wastewater.However,as one of the common dye contaminants,methyl orange(MO) has very weak affinity to metallic surfaces and is difficult to be detected by SERS at low concentrations.Therefore,a new type of SERS substrate with Ag nanoparticle monolayer functionalized by mono-6-deoxy-6-thio-β-cyclodextrin(β-CD-SH) was prepared to efficiently capture and detect MO in wastewater with a limit of detection of 5×10^-7 mol/L.The hydrophobic cavity of β-CD is responsible for the efficient trap and enrichment of MO on the Ag NPs surface,achieving a strong SERS signal of MO at low concentrations and at different pH values.This study provides new insight into designing a well-performed adsorbent for the capture and detection of organic contaminants.     

Ultrasensitive SERS Detection of TNT by Imprinting Molecular Recognition Using a New Type of Stable Substrate

We report herein a method for the ultra‐trace detection of TNT on p‐aminothiophenol‐functionalized silver nanoparticles coated on silver molybdate nanowires based on surface‐enhanced Raman scattering (SERS). The method relies on π‐donor–acceptor interactions between the π‐acceptor TNT and the π‐donor p,p′‐dimercaptoazobenzene (DMAB), with the latter serving to cross‐link the silver nanoparticles deposited on the silver molybdate nanowires. This system presents optimal imprint molecule contours, with the DMAB forming imprint molecule sites that constitute SERS “hot spots”. Anchoring of the TNT analyte at these sites leads to a pronounced intensification of its Raman emission. We demonstrate that TNT concentrations as low as 10^?12 M can be accurately detected using the described SERS assay. Most impressively, acting as a new type of SERS substrate, the silver/silver molybdate nanowires complex can yield new silver nanoparticles during the detection process, which makes the Raman signals very stable. A detailed mechanism for the observed SERS intensity change is discussed. Our experiments show that TNT can be detected quickly and accurately with ultra‐high sensitivity, selectivity, reusability, and stability. The results reported herein may not only lead to many applications in SERS techniques, but might also form the basis of a new concept for a molecular imprinting strategy.     

可固定性荧光探针:线粒体成像的可靠工具

线粒体是一种具有双层膜结构的细胞器,参与细胞新陈代谢过程的能量循环以及离子平衡过程,在细胞生理过程中具有极其重要的意义。一些小分子荧光染料/探针结构中带有正电荷,因受到线粒体内膜负电势的牵引而标记在线粒体上,为研究线粒体的形态或功能提供了重要的可视化成像工具。然而,大多数线粒体染料/探针对线粒体的靶向标记稳定性仍不够理想,因为线粒体电势处于不断的动态变化中,当电势降低时,对染料的亲和力相应降低。尤其在病理条件下(比如细胞凋亡)细胞代谢受到阻滞时,线粒体膜电势显著降低,阳离子染料将扩散离开线粒体,造成非特异性荧光。最近,Kim团队和本人课题组提出可固定线粒体探针的新概念,用活性基团将荧光分子探针通过共价键固定在线粒体中,开发了稳定靶向线粒体中的定量探测微环境p H值、粘度、膜电势荧光探针。我们认为,对于追踪和探测具有高度动态变化特性的线粒体而言,开发可固定的线粒体荧光分子探针是必然趋势,因此本文进行评述和展望。     

Microarray‐Based Detection of Dye‐Labeled DNA by SERRS Using Particles Formed by Enzymatic Silver Deposition

The growing interest in DNA diagnostics is addressed today by microarrays with fluoresence detection. In our approach, we utilize spatially defined arrays of short oligonucleotides on a modified glass surface. Surface enhanced resonance Raman scattering (SERRS) is used to obtain molecularly specific spectra of the Raman‐active dye‐labeled DNA. Nanoparticles produced by enzymatic silver deposition are used as SERS‐active substrate. They grow directly on the modified oligonucleotides and only in the spatially defined areas on the chip. Furthermore, they potentially offer several advantages for SERS detection. The nanoparticles are characterized and their ability for use as SERS‐ and SERRS‐active substrate is estimated. Three different Raman‐active dyes are investigated for their potential for involvement in sequence specific DNA analysis.     

A “Schizophotonic” All‐In‐One Nanoparticle Coating for Multiplexed SE(R)RS Biomedical Imaging

SERS nanoprobes for in vivo biomedical applications require high quantum yield, long circulation times, and maximum colloidal stability. Traditional synthetic routes require high metal–dye affinities and are challenged by unfavorable electrostatic interactions and limited scalability. We report the synthesis of a new near‐IR active poly(N‐(2‐hydroxypropyl) methacrylamide) (pHPMA). The integration of various SERS reporters into a biocompatible polymeric surface coating allows for controlled dye incorporation, high colloidal stability, and optimized in vivo circulation times. This technique allows the synthesis of very small (<20 nm) SERS probes, which is crucial for the design of excretable and thus highly translatable imaging agents. Depending on their size, the “schizophotonic” nanoparticles can emit both SERS and fluorescence. We demonstrate the capability of this all‐in‐one gold surface coating and SERS reporter for multiplexed lymph‐node imaging.     

Controlled Grafting Expansion Microscopy

Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.     

Selective surface-enhanced fluorescence and dye aggregation with layer-by-layer film substrates

One of the avenues for the development of the analytical applications of surface enhanced spectroscopy is the engineering of enhancing substrates that would be selective and target specific. In the present report, the proof of this concept is demonstrated using the layer-by-layer (LbL) technique to fabricate portable selective substrates containing metal nanoparticles which can provide surface enhanced fluorescence (SEF) or surface enhanced Raman scattering (SERS). The selectivity to ionic species is attained by adding metal-free top layers of polymer electrolytes to an LbL SERS enhancing substrate. In addition, it was observed that the surface charge of the top layer determines the dye aggregation, leading to the formation of adsorbed J or H aggregates.     

Preparation of Surface-enhanced Raman Scattering(SERS)-active Optical Fiber Sensor by Laser-induced Ag Deposition and Its Application in Bioidentification of Biotin/Avidin

A surface-enhanced Raman scattering(SERS) optical fiber sensor was prepared by the laser-induced deposition of Ag nanoparticle membrane on a silica optical fiber tip, which was applied to the real time SERS spectral monitoring on the biorecognition of biotin/avidin. The bioidentification of biotin/avidin was carried out through a indirect method, in which the bioidentification is based on the SERS response signal of a labeled dye(Atto610) after its fluorescence has been quenched totally by the deposited Ag nanoparticle membrane. By SERS monitoring the bioidentification process of biotin/avidin, it has been found that this recognition process is finished in 40 min. The lowest detection concentration of biotin is 1.0×10^-7 mg/mL. This research is promising in the application of immunoassays on line and in vivo.     

Label‐Free Detection of Glycan–Protein Interactions for Array Development by Surface‐Enhanced Raman Spectroscopy (SERS)

A glyco‐array platform has been developed, in which glycans are attached to plasmonic nanoparticles through strain‐promoted azide‐alkyne cycloaddition. Glycan–protein binding events can then be detected in a label‐free manner employing surface‐enhanced Raman spectroscopy (SERS). As proof of concept, we have analyzed the binding of Gal1, Gal3, and influenza hemagglutinins (HAs) to various glycans and demonstrated that binding partners can be identified with high confidence. The attraction of SERS for optical sensing is that it can provide unique spectral signatures for glycan–protein complexes, confirm identity through statistical validation, and minimizes false positive results common to indirect methods. Furthermore, SERS is very sensitive and has multiplexing capabilities thereby allowing the simultaneous detection of multiple analytes.     

Silica-void-gold nanoparticles: temporally stable surface-enhanced Raman scattering substrates

Reproducible detection of a target molecule is demonstrated using temporally stable solution-phase silica-void-gold nanoparticles and surface-enhanced Raman scattering (SERS). These composite nanostructures are homogeneous (diameter = 45 +/- 4 nm) and entrap single 13 nm gold nanoparticle cores inside porous silica membranes which prevent electromagnetic coupling and aggregation between adjacent nanoparticles. The optical properties of the gold nanoparticle cores and structural changes of the composite nanostructures are characterized using extinction spectroscopy and transmission electron microscopy, respectively, and both techniques are used to monitor the formation of the silica membrane. The resulting nanostructures exhibit temporally stable optical properties in the presence of salt and 2-naphthalenethiol. Similar SERS spectral features are observed when 2-naphthalenethiol is incubated with both bare and membrane-encapsulated gold nanoparticles. Disappearance of the S-H Raman vibrational band centered at 2566 cm(-1) with the composite nanoparticles indicates that the target molecule is binding directly to the metal surface. Furthermore, these nanostructures exhibit reproducible SERS signals for at least a 2 h period. This first demonstration of utilizing solution-phase silica-void-gold nanoparticles as reproducible SERS substrates will allow for future fundamental studies in understanding the mechanisms of SERS using solution-phase nanostructures as well as for applications that involve the direct and reproducible detection of biological and environmental molecules.     

Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer

Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.     

Ag nanoparticles prepared by laser photoreduction as substrates for in situ surface-enhanced Raman scattering analysis of dyes

In this work Ag nanoparticles (AgNP) with surface-enhanced Raman scattering (SERS) activity were prepared and immobilized by laser irradiation on a water/ solid interface where the aqueous phase contains the Ag+ cation and the solid surface is of hydrophilic nature (glass and cellulose). The so-prepared AgNP demonstrated a high SERS effectiveness in the detection of dispersed adsorbates such as the case of the anthraquinonic dye alizarin. The size and SERS effectiveness of AgNP increases with the irradiation time, the laser power, and the cation concentration. Laser-induced AgNP can be classified into two classes attending to the morphology: spherical and planar. The latter are formed after longer irradiation times, being more active regarding the SERS efficiency. Ag photoreduction can be employed for in situ detection of the dye alizarin, but when the dye is placed on a hydrophilic substrate. Even so, this in situ SERS technique could be attractive for analytical applications involving the in situ detection of the analyzed species, such as the case of dyes in artistical objects, textiles, foods, and surface analysis in general.     

Magnetic assistance highly sensitive protein assay based on surface-enhanced resonance Raman scattering

A simple and effective surface-enhanced Raman scattering (SERS)-based protocol for the detection of protein-small molecule interactions has been developed. We employed silver-coated magnetic particles (AgMNPs), which can provide high SERS activity as a protein carrier to capture a small molecule. Combining magnetic separation and the SERS method for protein detection, highly reproducible SERS spectra of a protein-small molecule complex can be obtained with high sensitivity. This time-saving method employs an external magnetic field to induce the AgMNPs to aggregate to increase the amount of atto610-biotin/avidin complex in a unit area with the SERS enhancement. Because of the contribution of the AgMNP aggregation to the SERS, this protocol has great potential for practical high-throughput detection of the protein-small molecule complex and the antigen-antibody immunocomplex.     

Synthesis of new, BODIPY-based sensors and labels

New, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye based thiol-reactive fluorescent label, fluorescent amino acid, and fluoroionophore compounds with 540-560 nm emission are described. Combination of a BODIPY dye with a nitronyl nitroxide or an imino nitroxide or a bifunctional pyrroline nitroxide furnished a nitric oxide, a redox sensitive molecule and a double (spin and fluorescence) label, respectively.