Raman multiplexers for alternative gene splicing

Nonfluorescent labels were used for an array-format multiplex detection of alternative splice junctions of breast cancer susceptibility gene 1 (BRCA1) by surface-enhanced Raman scattering (SERS). A four-plex detection scheme using nonfluorescent labels was demonstrated for DNA sequences specific to four BRCA1 alternative splice variants: Delta(11q) (the last 3309 nt deleted from exon 11), Delta(9, 10) (exon 9 and 10 deleted), Delta(5) (exon 5 deleted), and Delta(5q, 6) (the last 22 nt of exon 5 and the entire exon 6 is deleted). This is the first proof-of-concept study to apply SERS-based detection using nonfluorescent labels to investigate alternative gene splicing. Detection sensitivity of up to 1 fM was demonstrated for the Raman labels chosen to clearly discriminate the splice junctions via specific target identification. The proposed approach has the potential to become a highly sensitive and selective tool for comprehensive alternative splicing profiling of BRCA1 or other genes relevant to specific diseases.     

Surface-enhanced Raman scattering substrate based on a self-assembled monolayer for use in gene diagnostics

The development of surface-enhanced Raman scattering (SERS)-active substrates for cancer gene detection is described. The detection method uses Raman active dye-labeled DNA gene probes, self-assembled monolayers, and nanostructured metallic substrates as SERS-active platforms. The mercaptohexane-labeled single-stranded DNA (SH-(CH(2))(6)-ssDNA)/6-mercapto-1-hexanol system formed on a silver surface is characterized by atomic force microscopy. The surface-enhanced Raman gene (SERGen) probes developed in this study can be used to detect DNA targets via hybridization to complementary DNA probes. The probes do not require the use of radioactive labels and have a great potential to provide both sensitivity and selectivity. The effectiveness of this approach and its application in cancer gene diagnostics (BRCA1 breast cancer gene) are investigated.     

Graphene-based high-efficiency surface-enhanced Raman scattering-active platform for sensitive and multiplex DNA detection

We have developed a surface-enhanced Raman scattering (SERS)-active substrate based on gold nanoparticle-decorated chemical vapor deposition (CVD)-growth graphene and used it for multiplexing detection of DNA. Due to the combination of gold nanoparticles and graphene, the Raman signals of dye were dramatically enhanced by this novel substrate. With the gold nanoparticles, DNA capture probes could be easily assembled on the surface of graphene films which have a drawback to directly immobilize DNA. This platform exhibits extraordinarily high sensitivity and excellent specificity for DNA detection. A detection limit as low as 10 pM is obtained. Importantly, two different DNA targets could be detected simultaneously on the same substrate just using one light source.     

Spectroscopic tags using dye-embedded nanoparticles and surface-enhanced Raman scattering

Surface-enhanced Raman scattering is capable of providing rich vibrational information at the level of single molecules and single nanoparticles, but the practical applications of this enormous enhancement effect are still a challenge. Here we report a new class of dye-embedded core-shell nanoparticles that are highly efficient for surface Raman enhancement and could be used as spectroscopic tags for multiplexed detection and spectroscopy. The core-shell particles contain a metallic core for optical enhancement, a reporter molecule for spectroscopic signature, and an encapsulating silica shell for protection and conjugation. A surprising finding is that organic molecules with an isothiocyanate (-N=C=S) group or multiple sulfur atoms are compatible with silica encapsulation. In comparison with fluorescent dyes and quantum dots, enhanced Raman probes contain a built-in mechanism for signal amplification and provide rich spectroscopic information under ambient experimental conditions.     

A SERS‐Assisted 3D Barcode Chip for High‐Throughput Biosensing

A surface enhanced Raman scattering (SERS)‐assisted 3D barcode chip has been developed for high‐throughput biosensing. The 3D barcode is realized through joint 2D spatial encoding with the Raman spectroscopic encoding, which stores the SERS fingerprint information in the format of a 2D array. Here, the concept of SERS‐assisted 3D barcode is demonstrated through multiplex immunoassay, where simultaneous detection of multiple targets in different samples has been achieved using a microfluidic platform. First, multiple proteins in different samples are spatially separated using a microfluidic patterned antibody barcode substrate, forming a 2D hybridization array. Then the SERS probes are used to identify and quantify the proteins. As different SERS probes are labeled with different Raman reporters, they could be employed as “SERS tags” to incorporate spectroscopic information into the 3D barcode. In this 3D barcode, the 2D spatial information helps to differentiate the samples and targets while the SERS information allows quantitative multiplex detection. It is found that the SERS‐assisted 3D barcode chip can not only accomplish one‐step multiplex detection within 30 min but also achieve an ultrasensitivity down to 10 fg mL^?1 (≈70 aM), which is expected to provide a promising tool for high‐throughput biomedical applications.     

Surface-enhanced Raman scattering detection of DNA derived from the west nile virus genome using magnetic capture of Raman-active gold nanoparticles

A model paramagnetic nanoparticle (MNP) assay is demonstrated for surface-enhanced Raman scattering (SERS) detection of DNA oligonucleotides derived from the West Nile virus (WNV) genome. Detection is based on the capture of WNV target sequences by hybridization with complementary oligonucleotide probes covalently linked to fabricated MNPs and Raman reporter tag-conjugated gold nanoparticles (GNPs) and the subsequent removal of GNP-WNV target sequence-MNP hybridization complexes from solution by an externally applied magnetic source. Laser excitation of the pelleted material provided a signature SERS spectrum which is diagnostic for the reporter, 5,5'-dithiobis(succinimidy-2-nitrobenzoate) (DSNB), and restricted to hybridization reactions containing WNV target sequences. Hybridizations containing dilutions of the target oligonucleotide were characterized by a reduction in the intensification of the spectral peaks accorded to the SERS signaling of DSNB, and the limit of detection for target sequence in buffer was 10 pM. Due to the short hybridization times required to conduct the assay and ease with which reproducible Raman spectra can be acquired, the assay is amenable to adaptation within a portable, user-friendly Raman detection platform for nucleic acids.     

Au@pNIPAM SERRS Tags for Multiplex Immunophenotyping Cellular Receptors and Imaging Tumor Cells

Detection technologies employing optically encoded particles have gained much interest toward clinical diagnostics and drug discovery, but the portfolio of available systems is still limited. The fabrication and characterization of highly stable surface‐enhanced resonance Raman scattering (SERRS)‐encoded colloids for the identification and imaging of proteins expressed in cells are reported. These plasmonic nanostructures are made of gold octahedra coated with poly(N‐isopropylacrylamide) microgels and can be readily encoded with Raman active dyes while retaining high colloidal stability in biofluids. A layer‐by‐layer polyelectrolyte coating is used to seal the outer surface of the encoded particles and to provide a reactive surface for covalent conjugation with antibodies. The targeted multiplexing capabilities of the SERRS tags are demonstrated by the simultaneous detection and imaging of three tumor‐associated surface biomarkers: epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and homing cell adhesion molecule (CD44) by SERRS spectroscopy. The plasmonic microgels are able to discriminate tumor A431 (EGFR+/EpCAM+/CD44+) and nontumor 3T3 2.2 (EGFR?/EpCAM?/CD44+) cells while cocultured in vitro.     

Competitive Reaction Pathway for Site‐Selective Conjugation of Raman Dyes to Hotspots on Gold Nanorods for Greatly Enhanced SERS Performance

Common methods to prepare SERS (surface‐enhanced Raman scattering) probes rely on random conjugation of Raman dyes onto metal nanostructures, but most of the Raman dyes are not located at Raman‐intense electromagnetic hotspots thus not contributing to SERS enhancement substantially. Herein, a competitive reaction between transverse gold overgrowth and dye conjugation is described to achieve site selective conjugation of Raman dyes to the hotspots (ends) on gold nanorods (GNRs). The preferential overgrowth on the nanorod side surface creates a barrier to prevent the Raman dyes from binding to the side surface except the ends of the GNRs, where the highest SERS enhancement factors are expected. The SERS enhancement observed from this special structure is dozens of times larger than that from conjugates synthesized by conventional methods. This simple and powerful strategy to prepare SERS probes can be extended to different anisotropic metal nanostructures with electromagnetic hotspots and has immense potential in in‐depth SERS‐based biological imaging and single‐molecule detection.     

Composite organic-inorganic nanoparticles (COINs) with chemically encoded optical signatures

To obtain a coding system for multiplex detection, we have developed a method to synthesize a new type of nanomaterial called composite organic-inorganic nanoparticles (COINs). The method allows the incorporation of a broad range of organic compounds into COINs to produce surface enhanced Raman scattering (SERS)-like spectra that are richer in variety than fluorescence-based signatures. Preliminary data suggest that COINs can be used as Raman tags for multiplex and ultrasensitive detection of biomolecules.     

Characterization and cytocompatibility of carbon layers prepared by photo-induced chemical vapor deposition

We studied the preparation of the carbon layers on polytetrafluoroethylene (PTFE) by chemical vapor deposition from acetylene induced by UV-excimer lamp. Their surface properties and chemical structure were characterized by Atomic Force Microscopy, Scanning Electron Microscopy, Raman spectroscopy, Rutherford Backscattering Spectrometry, Elastic Recoil Detection Analysis, X-ray Induced Photoelectron Spectroscopy and others. The deposited layers could be characterized as hydrogenated amorphous carbon (a-C:H) containing additional oxygenic structures and conjugated double bonds. The cytocompatibility of the samples was tested with using of human umbilical endothelial cells (HUVEC). In comparison with pristine PTFE, the deposition resulted in drastically increased adhesion and proliferation of HUVEC.     

Rational design of fiber-optic probes for visible and pulsed-ultraviolet resonance Raman spectroscopy

We investigated the performance of fiber-optic resonance Raman probes with a series of experiments to determine the working curves of such probes using model analytes and to investigate the effects of absorbing media. A computer model designed to simulate these experiments is presented, and numerical results are found to be in agreement with the experimental data. Design considerations resulting from these studies are discussed, and novel designs for overcoming problems of coupling efficiency, damage threshold, and sensitivity in absorbing samples are presented. These findings are applied to the design of fiber-optic probes for ultraviolet resonance Raman spectroscopy involving nanosecond pulsed-ultraviolet excitation (225 and 266 nm). These probes have been used to collect what is, to our knowledge, the first reported fiber-optic-linked ultraviolet resonance Raman spectra of tryptophan and DNA.     

Polymerase chain reaction coupling with magnetic nanoparticles-based biotin-avidin system for amplification of chemiluminescent detection signals of nucleic acid

A novel method was established through the detection of chemiluminescent signals of nucleic acid hybridization based on magnetic nanoparticles (MNPs) and PCR. 5' amino- modified specific probes were immobilized on the surface of silanized MNPs by Schiff reaction between amino and aldehyde group. The probes were used to capture the synthetic biotin-dUTP-labeled DNA fragments which were obtained by polymerase chain reaction (PCR). Then these complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)- 4-methoxy -4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The concentration of probes which were immobilized on the surface of MNPs was studied, how to reduce the adsorption of SA-AP on the surface of MNPs was also researched. It was shown that 12.5 pmol of probes were immobilized on 1 mg of MNPs. Aldehyde-MNPs modified with probes could adsorb SA-AP, affecting the sensitivity of chemiluminescene consequently. Reduction of aldehyde group by sodium borohydride and blocking the bare position of MNPs with bovine serum albumin (BSA) could decrease the background of chemiluminescence, and this method has good specificity in detection of chloramphenicol acetyltransferase (CAT) gene.     

Detecting genomic aberrations by fluorescence in situ hybridization with quantum dots-labeled probes

Detection of genomic alterations of cancer genes by fluorescent in situ hybridization (FISH) will provide important information for cancer diagnosis and therapy. To effectively and reliably detect the genomic changes, we prepared novel FISH probes by directly conjugating genomic DNA of genes to semiconductor quantum dot fluorophores (QDs). The generated QD-genomic probes are substantially more photostable than the probes labeled with organic dye and show high intensity in both metaphase and interphase cell. The directly labeling probes allow detection of genomic targets in a fast and simple FISH procedure with high sensitivity and specificity. Furthermore, application of the QD-genomic probes in lung cancer specimens can reliably visualize gene amplification in cancer cells. These results suggest that the QD-FISH probes may offer an effective approach to analyze cancer-related genomic aberrations in basic research and clinical applications.     

Studies of molecular recognition mechanism in supramolecular system of ruthenium(II) polypyridyl complexes and DNA

The recent studies on supramolecular system assembled by Ru(II) polypyridyl complexes and B-form double-stranded DNA is reviewed. These complexes have been studied as DNA spectroscopic tags and structural probes. The studies focus on the recognition parameters of these complexes binding to DNA, including the influence of the shape and size of intercalative ligand, the ancillary ligands and the effects of hydrogen bonding. Enantioselectivities of these complexes binding to DNA are also briefly discussed.     

A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA

This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.     

A Raman waveguide detector for liquid chromatography

A novel real-time liquid core Raman waveguide detector designed for liquid chromatographic applications is described. The Raman waveguide detector provides enhanced selectivity over typical high-performance liquid chromatography (HPLC) detectors. The waveguide detector also greatly improves the sensitivity of a typical Raman measurement without resorting to surface enhancement or resonance approaches and is compatible with the typical peak width volumes eluted by microbore and minibore HPLC (packed 1-2-mm-i.d. columns). Detection limit enhancements of over 1000-fold have been achieved for the measurement of alcohols in the aqueous phase with the Raman cell utilizing liquid core waveguide technology. The liquid core waveguides demonstrated in this study were constructed using Teflon AF 2400 tubing with a refractive index of 1.29. The low refractive index of the polymer material allowed HPLC separations with Raman detection to be performed with an aqueous mobile phase. A calibration curve for aqueous solutions of 2-propanol was generated and a limit of detection (LOD) of 2 ppm was determined. The Raman waveguide detector is demonstrated for the HPLC analysis of alcohol test mixtures, with LODs in the low-ppm range at the detector. By coupling the temporal separation achieved by HPLC with the vibrational information gleaned from Raman detection, an information-rich multivariate data matrix is obtained that can be deconvoluted to provide chemical speciation even when the HPLC resolution is poor. In this paper, we will discuss the physical and optical design of the Raman waveguide detector and the demonstration of the detector for HPLC detection.     

Uniform gold nanoarray formed by controlled IP6 micelles for chemical mapping

A uniform Au nanoarray is successfully formed at an indium tin oxide (ITO) glass surface modified with well-distributed inositol hexakisphosphoric (IP(6)) micelle layers by controlling the pH of the medium at 10. When Rhodamine 6G (R6G) and 2-mercaptopyridine (2-MPy) are used as the Raman probes, the uniform Au nanoarray presents a sound surface enhanced Raman scattering (SERS) efficiency and a reproducible Raman signal in two dimensions. The relative standard deviation (RSD) of Raman intensities of R6G or 2-MPy on the uniform Au nanoarray recorded by point to point is less than 12%, which is beneficial to its application for chemical mapping or imaging. A case of Raman point-mapping for onion epidermis is demonstrated in the present work. A uniform IP(6)-Au nanoarray might be mass-produced by this protocol.     

C18-modified metal-colloid substrates for surface-enhanced Raman detection of trace-level polycyclic aromatic hydrocarbons in aqueous solution

Metal colloids immobilized on a glass support substrate are modified with a self-assembled alkylsilane (C18) layer to promote adsorption of polycyclic aromatic hydrocarbons from aqueous solutions. Detection of these compounds from low concentration solutions is accomplished by using surface-enhanced Raman scattering (SERS). SERS spectra of pyrene adsorbed to C18-modified immobilized silver colloids are dominated by Raman bands that are not consistent with pyrene and indicate that pyrene undergoes a chemical reaction at the surface. The origins of this surface product are investigated, and it is determined that silver and oxygen are required to form the product, whose Raman spectrum is consistent with oxidation to a quinone. When a C18-modified gold-colloid substrate is used, Raman scattering consistent with unreacted pyrene is observed. The adsorption and detection of pyrene adsorbed from low (2 ppb) concentration aqueous solutions onto C18-modified gold-colloid substrates is reported; naphthalene and phenanthrene are detected at approximately 5 ppb. Adsorption kinetics are rapid (<5 min), and the concentration-dependent SERS response is consistent with a Langmuir isotherm.     

Porous Au–Ag Nanoparticles from Galvanic Replacement Applied as Single-Particle SERS Probe for Quantitative Monitoring

Plasmonic nanostructures have raised the interest of biomedical applications of surface-enhanced Raman scattering (SERS). To improve the enhancement and produce sensitive SERS probes, porous Au–Ag alloy nanoparticles (NPs) are synthesized by dealloying Au–Ag alloy NP-precursors with Au or Ag core in aqueous colloidal environment through galvanic replacement reaction. The novel designed core–shell Au–Ag alloy NP-precursors facilitate controllable synthesis of porous nanostructure, and dealloying degree during the reaction has significant effect on structural and spectral properties of dealloyed porous NPs. Narrow-dispersed dealloyed NPs are obtained using NPs of Au/Ag ratio from 10/90 to 40/60 with Au and Ag core to produce solid core@porous shell and porous nanoshells, having rough surface, hollowness, and porosity around 30–60%. The clean nanostructure from colloidal synthesis exhibits a redshifted plasmon peak up to near-infrared region, and the large accessible surface induces highly localized surface plasmon resonance and generates robust SERS activity. Thus, the porous NPs produce intensely enhanced Raman signal up to 68-fold higher than 100 nm AuNP enhancement at single-particle level, and the estimated Raman enhancement around 7800, showing the potential for highly sensitive SERS probes. The single-particle SERS probes are effectively demonstrated in quantitative monitoring of anticancer drug Doxorubicin release.     

Shape-dependent surface-enhanced Raman scattering in gold-Raman probe-silica sandwiched nanoparticles for biocompatible applications

To meet the requirement of Raman probes (labels) for biocompatible applications, a synthetic approach has been developed to sandwich the Raman-probe (malachite green isothiocyanate, MGITC) molecules between the gold core and the silica shell in gold-SiO? composite nanoparticles. The gold-MGITC-SiO? sandwiched structure not only prevents the Raman probe from leaking out but also improves the solubility of the nanoparticles in organic solvents and in aqueous solutions even with high ionic strength. To amplify the Raman signal, three types of core, gold nanospheres, nanorods and nanostars, have been chosen as the substrates of the Raman probe. The effect of the core shape on the surface-enhanced Raman scattering (SERS) has been investigated. The colloidal nanostars showed the highest SERS enhancement factor while the nanospheres possessed the lowest SERS activity under excitation with 532 and 785 nm lasers. Three-dimensional finite-difference time domain (FDTD) simulation showed significant differences in the local electromagnetic field distributions surrounding the nanospheres, nanorods, and nanostars, which were induced by the localized surface plasmon resonance (LSPR). The electromagnetic field was enhanced remarkably around the two ends of the nanorods and around the sharp tips of the nanostars. This local electromagnetic enhancement made the dominant contribution to the SERS enhancement. Both the experiments and the simulation revealed the order nanostars > nanorods > nanospheres in terms of the enhancement factor. Finally, the biological application of the nanostar-MGITC-SiO? nanoparticles has been demonstrated in the monitoring of DNA hybridization. In short, the gold–MGITC-SiO? sandwiched nanoparticles can be used as a Raman probe that features high sensitivity, good water solubility and stability, low-background fluorescence, and the absence of photobleaching for future biological applications.