Surface-enhanced Raman spectrometry on a silver-coated filter paper substrate

The production, morphology, and optical properties of silver-coated filter paper as a new active substrate for surface-enhanced Raman spectrometry (SERS) are reported. The procedure involves a simple, one-stage reduction of filter-paper-supported silver nitrate with sodium tetrahydroborate. Intense and reproducible spectra of a variety of ionic and molecular adsorbates including p-aminobenzoic acid, acridine, 9-aminoacridine, 2-aminoanthracene, 6-nitroquinoline and 5-aminoquinoline are obtained. Simplicity, low cost, speed and availability are salient features of the substrate, which extend the applicability of the SERS technique to more practical situations.     

Investigation of chemically modified barium titanate beads as surface-enhanced Raman scattering (SERS) active substrates for the detection of benzene thiol, 1,2-benzene dithiol, and rhodamine 6G

SERS active surfaces were prepared by depositing silver films using Tollen's reaction on to barium titanate beads. The SERS activity of the resulting surfaces was probed using two thiols (benzene thiol and 1,2-benzene dithiol) and rhodamine 6G. The intensity of the SERS signal for the three analytes was investigated as a function of silver deposition time. The results indicate that the SERS intensity increased with increasing thickness of the silver film until a maximum signal intensity was achieved; additional silver deposition resulted in a decrease in the SERS intensity for all of the studied molecules. SEM measurement of the Ag coated barium titanate beads, as a function of silver deposition time, indicate that maximum SERS intensity corresponded with the formation of atomic scale islands of silver nanoparticles. Complete silver coverage of the beads resulted in a decreased SERS signal and the most intense SERS signals were observed at deposition times of 30 min for the thiols and 20 min for rhodamine 6G.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

A Prussian blue-based flow-through renewable surface optosensor for analysis of ascorbic acid

Ascorbic acid is determined by a simple Bead Injection Spectroscopy–Flow Injection Analysis (BIS–FIA) system with spectrophotometric detection. The sensor is based on the decrease of absorbance obtained (720 nm) when Prussian blue (PB) is reduced by ascorbic acid. Commercial available flow-cell (Hellma 138-OS) is used and an appropriate volume of homogeneous bead suspension of Sephadex QAE A-25 was injected to fill this flow-cell for each measurement. The chromogenic reagent (PB) is injected into the carrier and immobilized on beads. When sample is injected, reaching the bead surface where PB is sorbed, ascorbic acid converts it to Prussian white form, which is transparent, producing the discoloration of the detection zone. At the end of the analysis, beads are discarded by reversing the flow and instantaneously transported out of the system.The calibration graph was linear over the range 5.1×10⁻⁶–6.8×10⁻⁵ M. The detection limit and RSD (%) were 4.5×10⁻⁷ M and 5.0%, respectively, using 800 μl of sample volume. This method is highly selective in the presence of other species that are normally encountered with the analyte. The sensor was applied satisfactorily to the determination ascorbic acid in fruit juices, pharmaceuticals, sweets and conservative liquids.     

Surface-enhanced Raman spectroscopic-encoded beads for multiplex immunoassay

A new type of encoded bead, which uses surface-enhanced Raman scattering (SERS), is described for multiplex immunoassays. Silver nanoparticles were embedded in sulfonated polystyrene (PS) beads via a polyol method, and they were used as SERS-active substrates. Raman-label organic compounds such as 4-methylbenzenethiol (4-MT), 2-naphthalenethiol (2-NT), and benzenethiol (BT) were then adsorbed onto the silver nanoparticles in the sulfonated PS bead. Although only three kinds of encoding have been demonstrated here, various combinations of these Raman-label organic compounds have the potential to give a large number of tags. The Raman-label-incorporated particles were then coated with a silica shell using tetraethoxyorthosilicate (TEOS) for chemical stability and biocompatibility. The resulting beads showed unique and intense Raman signals for the labeled organic compounds. We demonstrated that SERS-encoded beads could be used for multiplex detection with a model using streptavidin and p53. In our system, the binding event of target molecules and the type of ligand can be simultaneously recognized by Raman spectroscopy using a single laser-line excitation (514.5 nm).     

Immunoassay using probe-labelling immunogold nanoparticles with silver staining enhancement via surface-enhanced Raman scattering

This paper reports a novel immunoassay based on surface-enhanced Raman scattering (SERS) and immunogold labelling with silver staining enhancement. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule (e.g., 4-mercaptobenzoic acid) and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecule. All the self-assembled steps were subjected to the measurements of ultraviolet-visible (UV-vis) spectra to monitor the formation of a sandwich structure onto a substrate. The immunoassay was performed by a sandwich structure consisting of three layers. The first layer was composed of immobilized antibody molecules of mouse polyclonal antibody against Hepatitis B virus surface antigen (PAb) on a silicon or quartz substrate. The second layer was the complementary Hepatitis B virus surface antigen (Antigen) molecules captured by PAb on the substrate. The third layer was composed of the probe-labelling immunogold nanoparticles, which were modified by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and 4-mercaptobenzoic acid (MBA) as the Raman-active probe on the surface of gold colloids. After silver staining enhancement, the antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS signal at 1585 cm(-1) due to the [small nu](8a) aromatic ring vibration of MBA versus the concentration of analyte (Antigen) was obtained and the non-optimized detection limit for the Hepatitis B virus surface antigen was found to be as low as 0.5 [micro sign]g mL(-1).     

The optimisation of facile substrates for surface enhanced Raman scattering through galvanic replacement of silver onto copper

A fast and cost-effective approach for the synthesis of substrates used in surface enhanced Raman scattering (SERS) has been developed using galvanic displacement. Deposition of silver onto commercially available Cu foil has resulted in the formation of multiple hierarchical structures, whose morphology show dependence on deposition time and temperature. Analysis of the surface structure by scanning electron microscopy revealed that the more complex silver structures correlated well with increased deposition time and temperature. Using Rhodamine 6G (R6G) as a model Raman probe it was also possible to relate the substrate morphology directly with subsequent SERS intensity from the R6G analyte as well as the reproducibility across a total of 15 replicate Raman maps (20 × 20 pixels) consisting of 400 spectra at a R6G concentration of 10(-4) M. The substrate with the highest reproducibility was then used to explore the limit of detection and this compared very favourably with colloidal-based SERS assessments of the same analyte.     

Surface-enhanced Raman spectroscopy based on conical holed enhancing substrates

In this contribution, surface-enhanced Raman spectroscopy (SERS) based on conical holed glass substrates deposited with silver colloids was reported for the first time. It combines the advantages of both dry SERS assays based on plane films deposited with silver colloids and wet SERS assays utilizing cuvettes or capillary tubes. Compared with plane glass substrates deposited with silver colloids, the conical holed glass substrates deposited with silver colloids exhibited five-to ten-folds of increase in the rate of signal enhancement, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte of interest. The conical holed glass substrates in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy (MEM_SERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4% and a limit of detection of about 0.02 μM using a portable i-Raman 785H spectrometer. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEM_SERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc.     

一种新的DNA探针-2-(N-二茂铁酰氨丙氧基)-6-氯-9-氨基吖啶的合成与性质

本文合成了一种含二茂铁侧基的DNA荧光探针-2-(N-二茂铁酰氨丙氧基)-6-氯-9-氨基吖啶, 并对该化合物的紫外可见光谱, 荧光光谱, NMR谱以及它和小牛胸腺DNA的作用与二茂铁侧基的吖啶作了比较和讨论。     

On-column silver substrate synthesis and surface-enhanced Raman detection in capillary electrophoresis

A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A ∼50-μm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-μm inner diameter CE capillary window or in a flow cell consisting of a 250-μm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20× objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25–25 ppm concentrations, by using 1.4–3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.     

In situ laser-induced photochemical silver substrate synthesis and sequential SERS detection in a flow cell

A new, simple, and effective approach for multianalyte sequential surface-enhanced Raman scattering (SERS) detection in a flow cell is reported. The silver substrate was prepared in situ by laser-induced photochemical synthesis. By focusing the laser on the 320 μm inner diameter glass capillary at 0.5 ml/min continuous flow of 1 mM silver nitrate and 10 mM sodium citrate mixture, a SERS active silver spot on the inner wall of the glass capillary was prepared in a few seconds. The test analytes, dacarbazine, 4-(2-pyridylazo)resorcinol (PAR) complex with Cu(II), and amoxicillin, were sequentially injected into the flow cell. Each analyte was adsorbed to the silver surface, enabling the recording of high intensity SERS spectra even at 2 s integration times, followed by desorption from the silver surface and being washed away from the capillary. Before and after each analyte passed the detection window, citrate background spectra were recorded, and thus, no “memory effects” perturbed the SERS detection. A good reproducibility of the SERS spectra obtained under flow conditions was observed. The laser-induced photochemically synthesized silver substrate enables high Raman enhancement, is characterized by fast preparation with a high success rate, and represents a valuable alternative for silver colloids as SERS substrate in flow approaches.     

Unexpected reactivity of the 9-aminoacridine chromophore in guanidylation reactions

The 9-aminoacridine chromophore is an important building block of DNA-targeted chemotherapeutic agents. The success of 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea as a carrier group in cytotoxic platinum-intercalator conjugates prompted us to explore the synthesis of an analogous guanidine-functionalized acridine. In a successful effort to generate such a derivative, various methods of guanidylation were employed, which demonstrate that the acridine C9-N9 linkage is highly susceptible to electrophilic and nucleophilic attack. The newly established reactivities provide efficient pathways to novel cyclic and spirocyclic acridine derivatives.     

A Bead Injection System for Copper Determination

ABSTRACT

A bead injection system for copper determination is proposed. Chelex-100 resin beads are introduced in the system as a suspension that is trapped in the Jet Ring Cell. The passage of the sample zone by the beads promotes the sorption of Cu(II). When the colorimetric reagent (APDC) perfuses the beads it reacts with copper ions, forming a colored complex that is monitored at 436 nm. After the measurement, the spent beads are sent to waste and a new portion of fresh beads is trapped in the system. The bead injection system is versatile and can be used to concentrate analyte in different sample volumes, permitting determinations of a wide range of copper concentrations. The detection limit is 0.5 μg l^-1 with a 500 μl sample, and 1.2 μg l^-1 with a 100 μ1 sample.     

Synthesis of pyrene—acridine bis-intercalators and effects of binding to DNA

A bis-intercalating compound containing pyrene and 9-aminoacridine chromophores (N-(5-(1-pyrenyl)-pentyl)-6-(9-acridinylamino) hexylamide, I), was prepared and its interaction with double-stranded DNA was investigated. Homologous compounds in which the two chromophores were connected by a linear carbon chain (pentamethylene (II), tetramethylene (III) and methylene (IV)) were also prepared. In acetonitrile solutions of the free ligands, the presence of the proximal pyrene results in reduced acridine fluorescence relative to 9methylaminoacridine (9-MAA), and the degree of quenching increases with decreasing chain length. The quenching process is assigned to exothermic electron transfer from pyrene to the excited 9-aminoacridine (9-AA) chromophore. In the presence of DNA, the relative quenching order is reversed, and I and IV are quenched more strongly than II and III. From linear dichroism experiments, it is concluded that I binds by bis-intercalation of the pyrene and acridine moieties, III and IV undergo intercalation of the acridine chromophore and II binds by partial bis-intercalation at two contiguous sites.     

Synthesis of highly ordered AgNPs-coated silica photonic crystal beads for sensitive and reproducible 3D SERS substrates

3 D highly ordered silver nanoparticles(AgNPs) coated silica photonic crystal beads(Ag/SPCBs) were prepared and exploited as a novel surface enhanced Raman scattering(SERS) substrate.The monodisperse and size-controlled SPCBs were prepared via self-assembly of silica nanoparticles process using a simple microfluidic device.Then the Ag/SPCBs were easily obtained by in situ growth of AgNPs onto the NH₂-modified SPCBs.Field emitting scanning electron microscopy(SEM) and energy dispersive X-ray spectrometry(EDX) were used to characterize the Ag/SPCBs.The effect of silica nanoparticle size and AgNO₃ concentration on the SERS performance of the resultant Ag/SPCBs substrate were discussed in detail.The results indicate that the Ag/SPCBs have highest SERS signals when silica nanoparticle size is250 nm and AgNO₃ concentration is 0.8 mg/mL.Using malachite green(MG) as model analyte,the Ag/SPCBs substrate displayed a high sensitivity and a wide linear range for MG.The well-designed Ag/SPCBs show high uniformity and excellent reproducibility,and can be used as an effective SERS substrate for sensitive assay application.     

Effect of spermine conjugation on the interaction of acridine with alternating purine-pyrimidine oligodeoxyribonucleotides studied by CD, fluorescence and absorption spectroscopies

We studied by electronic spectroscopies the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N(1)-(acridin-9-yl)-1,16-diamino-4,8,13-triazahexadecane, which is a conjugated molecule formed by the covalent binding of spermine and 9-aminoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate, at different molar ratios, were studied by using electronic absorption, fluorescence emission and circular dichroism. Calculated association constants and fluorescence emission spectra showed that spermine conjugation induces sequence selectivity. The orientation of the intercalated acridine rings with respect to the oligonucleotide base planes was deduced from the electronic circular dichroism spectra. Evidence of the formation of spermine-induced aggregated structures, with potential applications to DNA packaging, gene therapy and anti-tumor therapy, was also achieved. Our data demonstrates that this spermine-acridine conjugate adds several specific characteristics provided by the polyamine moiety, as sequence selectivity, to the interesting properties of acridine derivatives.     

微加工芯片式流通池在顺序注射可更新表面反射光谱检测中的应用

提出了一种以湿法蚀刻技术制备的用于流动注射（FI）或顺序注射（SI）进样可更新表面检测的芯片式流通池,并将此流通池与SI系统及检测器相匹配,用于顺序注射可更新表面（SI－RST）反射光谱法检测。流通池由两片玻璃封接而成,流通池通道蚀刻在玻璃基片上。流通池通过多股双岔光纤分别与光源、检测器相耦合,以实现对微珠表面的反射光谱法检测。将此SI－RST反射光谱法检测系统用于对锌的检测,并测定人发试样中微量锌的含量。     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.     

Resolution of biparametric mixtures using bead injection spectroscopic flow-through renewable surface sensors.

A new, sensitive and simple bead injection spectroscopy-flow injection analysis (BIS-FIA) sensor with spectrophotometric detection, using a commercially available flow-cell, is described to the determination of biparametric mixtures. As an analytical model, the metallic mixture Cu(II) and Zn(II) has been chosen. The flow-cell (Hellma 138-OS) is filled by injecting in the flow system 300 microl of a homogeneous bead suspension of an anion exchanger gel (Sephadex QAE A-25) previously loaded with the chromogenic reagent 2-carboxyl-2-hydroxy-5-sulfoformazylbenzene (Zincon). A sequential reaction of Cu(II) and Zn(II) with Zincon to form two complexes is performed on the bead sensing support and the absorbance is monitored at 627 nm, after two successive injections from the mixture solution. The sample containing these metal ions is injected into the first carrier (deionized water, pH 5.9), and Cu(II) selectively reacts with Zincon on the beads, developing the analytical signal. Then, 600 microl of 2 M HCl is injected to decompose the complex, and the carrier solution is changed. At pH 11 (second carrier) both Cu(II) and Zn(II) react with the chromogenic reagent, the absorbance now corresponding to both analytes. The eluent is again injected to descompose both complexes. After three analyses the sensing bead surface is not regenerated. Then, beads are automatically discarded from the flow cell by reversal of the flow, and instantaneously transported out of the system. So the procedure exploits the combination of the concepts of flow-through renewable sensors with bead injection spectroscopy. Using a sample volume of 1000 microl, the calibration graph for Cu(II) is linear over the range 0.05 to 1 microg ml(-1) and for Zn(II) from 0.1 to 1.8 microg ml(-1) in the presence of each other. RSDs (%) lower than 5% are obtained for both analytes. The sensor is satisfactorily applied to individual determination or mixture resolution in waters, pharmaceuticals, soils and human hair samples.     

Stability of silver colloids as substrate for surface enhanced Raman spectroscopy detection of dipicolinic acid

Silver colloids have been commonly used as substrates for surface enhanced Raman spectroscopy (SERS). It has been shown that SERS requires partial aggregation of the silver colloids. This study evaluates factors affecting the aggregative state of the silver colloids such as the age of the silver colloids and the aggregation as a result of addition of the analyte. The silver colloids are obtained from the chemical reduction of silver nitrate by sodium borohydride. Further oxidation of the sodium borohydride solution at room temperature results in concentration changes of the resulting silver colloids. Methods of controlling the sodium borohydride depletion are presented in this paper. The analyte used is dipicolinic acid, a molecular signature of Bacillus spores.