银-过硫酸钾-钍试剂体系催化光度法测定痕量银

本文研究了在pH=4.5的HAc-NaAc介质中,以2,2’-联吡啶为活化剂,过硫酸钾氧化钍试剂催化动力学光度法测定痕量银的新方法。本法检测限为2.49×10-3μg/mL;相对标准偏差为3.3%(n=11);线性范围为0.004~0.048μg/mL。可用于测定黑白相纸液中的银和氯化银的溶度积。     

Simplified protocol for detection of protein-ligand interactions via surface-enhanced resonance Raman scattering and surface-enhanced fluorescence

A simple and effective protocol for detections of protein-protein and protein-small molecule interactions has been developed. After interactions between proteins and their corresponding ligands, we employed colloidal silver staining for producing active substrates for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Tetramethylrhodamine isothiocyanate (TRITC) and Atto610 were used for both Raman and fluorescent probes. We detected interactions between human IgG and TRITC-anti-human IgG, and those between avidin and Atto610-biotin by surface-enhanced resonance Raman scattering (SERRS) and SEF. The detection limits of the proposed SERRS-based method are comparable to those of the proposed SEF-based one, 0.9 pg/mL for anti-human IgG and 0.1 pg/mL for biotin. This protocol exploits several advantages of simplicity over other SERS and SEF-based related methods because of the protein staining-based strategy for silver nanoparticle assembling, high sensitivity from SERRS and SEF, and high stability in photostability comparing to fluorescence-based protein detections. Therefore, the proposed method for detection of protein-ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination.     

Facile synthesis of Ag dendrites on Al foil via galvanic replacement reaction with [Ag(NH3)2]Cl for ultrasensitive SERS detecting of biomolecules

Symmetric silver dendrites have been synthesized on commercial aluminum foil via galvanic replacement reaction with [Ag(NH₃)₂]Cl. This process is facile and environmentally friendly, without the use of any templates, surfactants or oxidants, and also avoiding the introduction of fluoride anions as a strong toxicity resulting in hypocalcemia. The products were characterized with scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution TEM (HRTEM) and X-ray diffraction (XRD). SEM characterizations and electrochemical measurements including an electrochemical direct current polarization method and OCP-t technique demonstrate that chloride has proven to be the key factor to the formation of well-defined dendritic shape. The as-prepared Ag dendrites are developed as a surface-enhanced Raman scattering (SERS)-active platform for detection of folic acid, DNA and RNA with well resolved bands and high Raman intensities. The detection concentration for the three biomolecules reaches the level of 10⁻¹² M, and thus the symmetric silver dendrites can potentially be employed as effective SERS sensors for label-free and ultrasensitive biomolecule detection.     

Analytical technique for label-free multi-protein detection based on Western blot and surface-enhanced Raman scattering

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.     

Surface-enhanced Raman scattering imaging of a single molecule on urchin-like silver nanowires

Urchin-like silver nanowires are prepared by reacting AgNO(3)(aq) with copper metal in the presence of cetyltrimethylammonium chloride and HNO(3)(aq) on a screen-printed carbon electrode at room temperature. The diameters of the nanowires are about 100 nm, and their lengths are up to 10 μm. Using Raman spectroscopy, the detection limit of Rhodamine 6G (R6G) on the urchin-like silver nanowire substrate can be as low as 10(-16) M, while the analytical enhancement factor is about 10(13). Raman mapping images confirm that a single R6G molecule on the substrate can be detected.     

Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers

We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules.     

Sensitive chemiluminescence determination of thirteen cephalosporin antibiotics with luminol-copper(II) reaction

A new chemiluminescence reaction, the luminol-Cu(2+) reaction, was investigated for the determination of thirteen (13) cephalosporin antibiotics, namely cefalexin, cefadroxil, cefradine, cefazolin sodium, cefaclor, cefuroxime axetil, cefotaxime sodium, cefoperazone sodium, ceftriaxone sodium, ceftazidime, cefetamet pivoxil hydrochloride, cefixime, and cefpodoxime. It was found that, without adding any special oxidant, strong chemiluminescent (CL) signal could be produced from the reaction of the alkaline luminol with the above-mentioned antibiotics in the presence of Cu(2+). The experimental conditions for the reaction were carefully optimized with flow-injection mode. The detection limits are 0.3 ng/mL cefalexin, 3 ng/mL cefadroxil, 0.3 ng/mL cefradine, 0.02 μg/mL cefazolin sodium, 0.8 ng/mL cefaclor, 0.02 μg/mL cefuroxime axetil, 5 ng/mL cefotaxime sodium, 0.02 μg/mL cefoperazone sodium, 0.8 ng/mL ceftriaxone sodium, 1 ng/mL ceftazidime, 0.08 ng/mL cefetamet pivoxil hydrochloride, 0.8 ng/mL cefixime, and 2 ng/mL cefpodoxime. The proposed method was validated by direct application to commercial formulations and spiked milk samples containing cefradine. A possible reaction mechanism is also discussed.     

Mesoporous Silica Nanoparticles Act as a Self‐Adjuvant for Ovalbumin Model Antigen in Mice

Immunization to the model protein antigen ovalbumin (OVA) is investigated using MCM‐41 mesoporous silica nanoparticles as a novel vaccine delivery vehicle and adjuvant system in mice. The effects of amino surface functionalization and adsorption time on OVA adsorption to nanoparticles are assessed. Amino‐functionalized MCM‐41 (AM‐41) shows an effect on the amount of OVA binding, with 2.5‐fold increase in binding capacity (72 mg OVA/g AM‐41) compared to nonfunctionalized MCM‐41 (29 mg OVA/g MCM‐41). Immunization studies in mice with a 10 μg dose of OVA adsorbed to AM‐41 elicits both antibody and cell‐mediated immune responses following three subcutaneous injections. Immunizations at a lower 2 μg dose of OVA adsorbed to AM‐41 particles results in an antibody response but not cell‐mediated immunity. The level of antibody responses following immunization with nanoformulations containing either 2 μg or 10 μg of OVA are only slightly lower than that in mice which receive 50 μg OVA adjuvanted with QuilA, a crude mixture of saponins extracted from the bark of the Quillaja saponaria Molina tree. This is a significant result, since it demonstrates that AM‐41 nanoparticles are self‐adjuvanting and elicit immune responses at reduced antigen doses in vivo compared to a conventional delivery system. Importantly, there are no local or systemic negative effects in animals injected with AM‐41. Histopathological studies of a range of tissue organs show no changes in histopathology of the animals receiving nanoparticles over a six week period. These results establish the biocompatible MCM‐41 silica nanoparticles as a new method for vaccine delivery which incorporates a self‐adjuvant effect.     

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 μg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.     

Sensitive and Versatile Detection of the Fouling Process and Fouling Propensity of Proteins on Polyvinylidene Fluoride Membranes via Surface-Enhanced Raman Spectroscopy

Membrane fouling is the major drawback of membrane-based technologies because it will lead to severe flux declines and the need to clean or replace the fouled membrane. A technique capable of early diagnosis, process monitoring, and evaluation of the role of different foulants playing in the fouling process is crucial for the fouling control. We develop surface-enhanced Raman spectroscopy (SERS) as a new and versatile tool to investigate the fouling process of protein on PVDF (polyvinylidene fluoride) membranes as well as the fouling propensity of three different proteins. We optimized the aggregation level and volume of SERS-active Ag sol and the spectra acquisition method combined with a statistical analysis method to ensure a high detection sensitivity, signal uniformity, and stability. We then used SERS for the early diagnosis of the fouling process and determining when the membrane pores would be blocked. The fouled area was visualized by a combination of the silver staining and Raman mapping. The fouling propensity of different proteins was studied by comparing the relative SERS band intensities of different proteins on a glass slide and after membrane filtration. Compared with fluorescence-based techniques, the narrow, well-resolved Raman band, especially the use of the same excitation line and laser power, endows SERS the ability to compare the fouling propensity in a very simple way.     

Detection of anions by normal Raman spectroscopy and surface-enhanced Raman spectroscopy of cationic-coated substrates

The use of normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) of cationic-coated silver and gold substrates to detect polyatomic anions in aqueous environments is examined. For normal Raman spectroscopy, using near-infrared excitation, linear concentration responses were observed. Detection limits varied from 84 ppm for perchlorate to 2600 ppm for phosphate. In general, detection limits in the ppb to ppm concentration range for the polyatomic anions were achieved using cationic-coated SERS substrates. Adsorption of the polyatomic anions on the cationic-coated SERS substrates was described by a Frumkin isotherm. The SERS technique could not be used to detect dichromate, as this anion reacted with the coatings to form thiol esters. A competitive complexation method was used to evaluate the interaction of chloride ion with the cationic coatings. Hydrogen bonding and pi-pi interactions play significant roles in the selectivity of the cationic coatings.     

Optical detection of chloramphenicol using molecularly imprinted polymers

A practical optical sensing system for the determination of chloramphenicol (CAP), utilizing molecularly imprinted polymers (MIPs) and HPLC, has been developed. The method is based on competitive displacement of a chloramphenicol-methyl red (CAP-MR) dye conjugate from specific binding cavities in an imprinted polymer by the analyte. The best of these polymers was obtained using (diethylamino)ethyl methacrylate as functional monomer at a monomer:template ratio of 2:1. HPLC with a mobile phase containing CAP-MR was used as the detection system, and injection of CAP and, to a lesser degree, thiamphenicol resulted in proportional displacement of the conjugate, which was detected at 460 nm. The detection system showed a linear response over a range of 3-1000 μg/mL and effectively detected CAP extracted from serum. This system offers a tailor-made, selective, and rapid method for CAP detection, is able to discriminate between similar molecules, and is effective below and above the therapeutic range (10-20 μg/mL serum, potentially toxic above 25 μg/mL). This technique is quite general and should enable the use of MIPs in a wide variety of applications involving the detection of families of molecules which possess a distinct arrangement of functional groups.     

Raman spectroscopic detection for perchlorate at low concentrations

Perchlorate (ClO4-) has recently emerged as a widespread environmental contaminant found in groundwater and surface water, and there is a great need for rapid detection and monitoring of this contaminant. In this study, we explore the use of surface-enhanced (SERS) and normal Raman spectroscopy for detecting ClO4- at low concentrations. We found that ClO4- is SERS active and, for the first time, were able to detect ClO4- at concentrations as low as 10(-6)-10(-7) M (or 10-100 microg/L) through the application of silver SERS substrates or selective sorbents such as bifunctional anion-exchange resins. The use of selective sorbents greatly enhanced the reproducibility and sensitivity of ClO4- detection by normal Raman spectroscopy. Further exploration and research may allow application of these techniques for in situ, real-time detection and monitoring of ClO4- in environmental samples at even lower concentrations.     

Ultrasensitive detection of 1,4-Bis(4-vinylpyridyl)phenylene in a small volume of low refractive index liquid by surface-enhanced Raman scattering-active light waveguide

This paper reports a novel surface-enhanced Raman scattering (SERS)-active light waveguide method for ultrasensitive detection of a sample dissolved in a small volume of low refractive index liquid. The SERS-active light waveguide demonstrated in this study was constructed via the light-guiding silica capillary. The surface of its inner wall was modified with SERS-active silver nanoparticles that can remarkably enhance Raman signals. The capillary with SERS-active modified layer was filled with the sample solution to form the SERS-active liquid core (LC) fiber. The incident laser beam travels through the waveguide in a totally reflective mode within the fiber wall and penetrates a small distance into the sample solution by the evanescent wave field. The Raman scattering of the analytes adsorbed onto the surface of the SERS-active modified layer can be excited by the laser beam and refracted into the fiber wall. Thus, a sample dissolved in low index liquid, e.g., methanol, can be quantitatively monitored by Raman spectroscopy and detection limit of its concentration is lower than 10(-9) mol/L.     

纳米银/聚乙烯醇复合物的生物合成及其对6种水产病原菌的抑菌活性

纳米银作为一种新型抑菌剂有望成为传统抑菌剂的替代品,制备稳定、高效、环保的新型纳米银抑菌产品成为当今的研究热点。本研究以葡萄籽提取液为还原剂和稳定剂,聚乙烯醇(PVA)为载体,采用一步法“绿色”生物合成出一种纳米银/聚乙烯醇复合物(AgNPs/PVA)。通过紫外-可见(UV-Vis)吸收光谱、透射电镜(TEM)、X射线衍射(XRD)等手段对合成产物进行了表征。结果表明银离子被葡萄籽提取物成功还原成纳米银并附着在PVA的表面,纳米银颗粒均匀,呈现单分散状态,粒径较小,平均粒径为14 nm左右。AgNPs/PVA对鳗弧菌、溶藻弧菌、副溶血弧菌、哈维氏弧菌、灿烂弧菌及点状气单胞菌等6种典型的水产病原菌均有显著的抑菌效果。以溶藻弧菌为指示菌,AgNPs/PVA的最小抑菌浓度(MIC)为1.1 μg/mL,最小杀菌浓度(MBC)为2.2 μg/mL。AgNPs/PVA的Zeta电位为?24.1 mV,表明纳米银颗粒间有很强的排斥力,为其稳定分散提供保障,后续实验证明制备的AgNPs/PVA具有良好的稳定性和热稳定性。以上研究结果表明,AgNPs/PVA复合材料在水产养殖病害防治中具有广阔的应用前景。      

Minimization of transition metal interferences with hydride generation techniques

A new hydride generator has been characterized for use with the acid-NaBH(4) hydride generation systems based on the insertion of a capillary tube into the sample introduction channel of a standard Meinhard nebulizer. The acidic sample and the tetrahydroborate solution are mixed at a merge point 1.5 cm from the end of the nebulizer orifice. Nebulization of the reaction solutions into a 0.7 mL tubular "spray chamber" follows a very short mixing time (less than 0.012 s) of the reagents. This approach permits 10?000 μg/mL Ni(2+) or Cr(3+) to be present in the sample solution without producing any interferences. Additionally, in the presence of Fe(3+) added as a "releasing agent", 5000 μg/mL Co(2+) or 160 μg/mL Cu(2+) can also be tolerated without interference. An 80 ± 2% generation efficiency is attained for the test element selenium. A detection limit of 6 μg/L (3σ(b)) is achieved with ICP-AES detection. Precision of replicate measurements at the 12 μg/L level varies from 5 to 12% relative standard deviation.     

Chitosan-coated anisotropic silver nanoparticles as a SERS substrate for single-molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a technique that has become widely used for identifying and providing structural information about molecular species in low concentration. There is an ongoing interest in finding optimum particle size, shape and spatial distribution for optimizing the SERS substrates and pushing the sensitivity toward the single-molecule detection limit. This work reports the design of a novel, biocompatible SERS substrate based on small clusters of anisotropic silver nanoparticles embedded in a film of chitosan biopolymer. The SERS efficiency of the biocompatible film is assessed by employing Raman imaging and spectroscopy of adenine, a significant biological molecule. By combining atomic force microscopy with SERS imaging we find that the chitosan matrix enables the formation of small clusters of silver nanoparticles, with junctions and gaps that greatly enhance the Raman intensities of the adsorbed molecules. The study demonstrates that chitosan-coated anisotropic silver nanoparticle clusters are sensitive enough to be implemented as effective plasmonic substrates for SERS detection of nonresonant analytes at the single-molecule level.     

Serum analysis method combining cellulose acetate membrane purification with surface‐enhanced Raman spectroscopy for non‐invasive HBV screening

A highly sensitive, non‐invasive, and rapid HBV (Hepatitis B virus) screening method combining membrane protein purification with silver nanoparticle‐based surface‐enhanced Raman scattering (SERS) spectroscopy was developed in this study. Reproducible serum protein SERS spectra were obtained from cellulose acetate membrane‐purified human serum from 94 HBV patients and 89 normal groups. Tentative assignments of serum protein SERS spectra showed that the HBV patients primarily led to specific biomedical changes of serum protein. Principal components analysis and linear discriminate analysis were introduced to analyse the obtained spectra, with the diagnostic sensitivity of 92.6% and specificity of 77.5% were achieved for differentiating HBV patients from normal groups.Inspec keywords: patient diagnosis, surface enhanced Raman scattering, proteins, biomembranes, principal component analysis, purification, silver, nanoparticles, nanomedicine, diseasesOther keywords: serum analysis method, cellulose acetate membrane purification, surface‐enhanced Raman spectroscopy, noninvasive HBV screening, rapid HBV screening method, Hepatitis B virus, membrane protein purification, silver nanoparticle‐based surface‐enhanced Raman scattering spectroscopy, reproducible serum protein SERS spectra, cellulose acetate membrane‐purified human serum, linear discriminate analysis, diagnostic sensitivity, HBV patient, principal components analysis     

Discrimination of bacteria and bacteriophages by Raman spectroscopy and surface-enhanced Raman spectroscopy

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 10(9) pfu/mL and 10(6) cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 10(3) fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.     

邻菲罗啉共振光散射光谱法测定痕量镉的研究

基于镉与KI、邻菲罗啉形成多元配合物导致体系共振光散射强度增强,并且随着Cd2+浓度的加大,共振光散射的强度逐渐增大,建立一种邻菲罗啉共振光散射光谱法测定痕量镉的新方法。室温下,采用邻菲罗啉,pH=7.0条件下,在λ=397.6 nm处,Cd2+的加入浓度与共振光散射强度呈良好的线性关系,方法线性范围为0.01～6.5μg/mL,相关系数r=0.999 1,检出限0.06μg/mL,样品加标回收率为96.0%～102.8%。该法用于实际样品废旧电池中痕量镉的分析,效果较好。