疏水性SERS基底检测应用研究进展

本文简要介绍了表面增强拉曼散射(SERS)的拉曼信号增强原理,常规SERS增强基底的研究应用进展;同时介绍了疏水性基底的定义及优势,并重点综述了疏水性SERS基底的分类及研究应用的进展;最后展望了疏水性SERS基底的研究方向和发展趋势。疏水性SERS基底的发展将有望为今后开展面向超低浓度的生化物质检测以及基于人体体液的疾病的灵敏、客观检测诊断提供新方法和新思路。     

Validation and characterization of uninhibited enzyme kinetics performed in multiwell plates

Several systematic errors may occur during the analysis of uninhibited enzyme kinetic data using commercially available multiwell plate reader software. A MATLAB program is developed to remove these systematic errors from the data analysis process for a single substrate-enzyme system conforming to Michaelis-Menten kinetics. Three experimental designs that may be used to validate a new enzyme preparation or assay methodology and to characterize an enzyme-substrate system, while capitalizing on the ability of multiwell plate readers to perform multiple reactions simultaneously, are also proposed. These experimental designs are used to (i) test for enzyme inactivation and the quality of data obtained from an enzyme assay using Selwyn's test, (ii) calculate the limit of detection of the enzyme assay, and (iii) calculate Km and Vm values. If replicates that reflect the overall error in performing a measurement are used, the latter two experiments may be performed with internal estimation of the error structure. The need to correct for the systematic errors discussed and the utility of the proposed experimental designs were confirmed by numerical simulation. The proposed experiments were conducted using recombinant inducible nitric oxide synthase preparations and the oxyhemoglobin assay.     

Multianalyte immunoassay with self-assembled addressable microparticle array on a chip

This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the microfabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment. An array template was photoresist-patterned with spatially discrete microwells in which an electrodeposited Ni-PTFE hydrophobic composite layer and a hydrophobic photo-adhesive coating were deposited. The particles, after biomaterial attachment and binding processes in bulk, were self-assembled randomly onto the lubricated bonding sites on the chip substrate, driven by a combination of magnetic, hydrophobic, and capillary interactions. The encoding symbol carried by the particles was used as the signature for the identification of each target/assay attached to the particle surface. We demonstrate here the utility of microfabricated-encoded particle arrays for conducting multianalyte immunoassays in a parallel fashion with the use of imaging detection.     

Fabrication of functionalized double-lamellar multifunctional envelope-type nanodevices using a microfluidic chip with a chaotic mixer array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>10(5)) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Green synthesis of silver nanoparticles with Dalbergia spinosa leaves and their applications in biological and catalytic activities

The phytosynthesis of silver nanoparticles (AgNPs) by Dalbergia spinosa leaves (DSL) in aqueous extract was investigated. AgNPs were characterized by UV–visible absorption spectroscopy (UV–vis), transmission electron microscopy (TEM) and Fourier transform infra red spectrophotometry (FTIR). The results showed that the increase in the initial extract concentration at room temperature increased the mean size and widened the size distribution of the AgNPs, leading to a red shift and broadening the surface plasmon resonance absorption (439 nm). The results showed that the reducing sugars and flavonoids were primarily responsible for the bioreduction of silver ions and that their reductive capability was promoted at 36 °C. TEM analysis showed that the AgNPs were nearly spherical in shape with an average size of 18 ± 4 nm. When evaluated for in vitro antioxidant activity by DPPH, NO, hydrogen peroxide radicals, reducing power and CUPRAC assay methods in addition to anti-inflammatory activity by HBRC method, the silver nanoparticles exhibited considerably enhanced antioxidant and anti-inflammatory activity at the test doses when compared with that of the standards and the plant extract. Finally, the antibacterial activity of the AgNPs against two Gram-positive bacteria and two Gram-negative bacteria showed moderate antibacterial activity when compared with the standard and the plant extract. The synthesized silver nanoparticles were also effective in the catalytic reduction of 4-nitrophenol (4-NP) into 4-aminophenol (4-AP).     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

A designed glycopeptide array for characterization of sugar-binding proteins toward a glycopeptide chip technology

For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as “protein fingerprints” (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies.     

The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites

FRISVAD, J. C, 1989. The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites. Fungal chemotaxonomy (that part dealing with secondary metabolites) has often been based on thin layer chromatography (TLC) and visual or UV inspection of separated spots, before and after different chemical treatments. The identity of a small proportion of the spots can be suggested based on known internal and external standards. In most chemotaxonomical studies it is impossible to isolate, purify and identify all secondary metabolites produced, due to restraints of time and resources. High performance liquid chromatography (HPLC) of fungal extracts may have some advantages over TLC, but the problems mentioned above remain. These problems have been approached by using an alkylphenone retention time index in a reversed phase HPLC system combined with the use of a diode array UV-VIS detector. High performance thin layer chromatography is used for further confirmation of identity of the secondary metabolites. A particular advantage of this method is that the number of biosynthetic families or groups ('chemosyndromes') can be detected, as biosynthetically related metabolites usually have the same chromophores and UV-VIS spectra. Results obtained from Penicillium, Aspergillus and Fusanum species have shown that each species produces 5 to 15 different biosynthetic families of secondaiy metabolites, indicating that good chromatography data may be sufficient to identify species in the three genera. The use of the technique is exemplified by data on Aspergillus and Talaromyces species.     

Bio-Plex悬浮芯片系统检测两种实验兔白介素基因的差异表达

目的采用液相悬浮芯片系统同时测定实验兔圆小囊中IL-1β、IL-1R1、IL=8、IL-8RA和IL=15各基因的表达情况,并对该方法进行评价。方法利用Affymetrix的Panomics QuantiGene Plex2．0Assay中bDNA信号放大和多磁珠分析技术,来同时检测两种实验兔圆小囊中多重mRNA并定量。建立实验兔免疫相关白介素基因的液相悬浮芯片检测方法。结果可同时检测IL-1β、IL=1R1、IL-8、IL=8RA和IL-15各基因的含量,并发现WHBE兔IL-15基因的相对表达量显著高于JW兔（P〈0．05）,IL-1R1基因的相对表达量显著高于JW兔（P〈0．01）,IL-8RA基因在WHBE兔中的相对表达量也高于JW兔（P〈0．05）。结论建立了实验兔白介素基因的液相悬浮芯片检测方法,WHBE兔的IL-15、IL-1R1和IL-8RA基因表达量较高,可能与WHBE兔独特的免疫学特性有关。     

Identification of hemorphins in a cathepsin D bovine hemoglobin hydrolysate by radioimmunoassay and photodiode array detections

Summary Morphinomimetic peptides have been purified from hemoglobin enzymatic hydrolysates and a significant amount of evidence has been accumulated indicating that the generation of these peptides (hemorphins) might occur in vivo. In order to investigate their putative physiological role and processing from hemoglobin in vivo, two methods were developed: a specific radioimmunoassay and a UV spectra comparison analysis. These methods were applied to a cathepsin D bovine hemoglobin hydrolysate and allowed the detection of two hemorphin-7 peptides. This observation supports the putative implication of cathepsin D in the in vivo release of hemorphins. Among the two methods used in this study, the immunological approach exhibits higher sensitivity and represents a useful method to investigate the in vivo role and physiological processing of hemorphins.     

Identification of hemorphins in a cathepsin D bovine hemoglobin hydrolysate by radioimmunoassay and photodiode array detections

Morphinomimetic peptides have been purified fromhemoglobin enzymatic hydrolysates and a significantamount of evidence has been accumulated indicatingthat the generation of these peptides (hemorphins)might occur in vivo. In order to investigatetheir putative physiological role and processing fromhemoglobin in vivo, two methods were developed:a specific radioimmunoassay and a UV spectracomparison analysis. These methods were applied to acathepsin D bovine hemoglobin hydrolysate and allowedthe detection of two hemorphin-7 peptides. Thisobservation supports the putative implication ofcathepsin D in the in vivo release ofhemorphins. Among the two methods used in this study,the immunological approach exhibits highersensitivity and represents a useful method toinvestigate the in vivo role and physiologicalprocessing of hemorphins.     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-1-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA ofp53 gene,N-ras gene andRb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

A new method of preparing fiber-optic DNA biosensor and its array for gene detection

A new method of preparing fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were first treated with poly-l-lysine, and then were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end. By assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe, the fiber-optic DNA biosensor array was well prepared. Hybridization of fluorescent- labeled cDNA of p53 gene, N-ras gene and Rb1 gene to the DNA array was monitored by CCD camera. A good result was achieved.     

Detection of tetanus-induced effects in linearly lined-up micropatterned neuronal networks: application of a multi-electrode array chip combined with agarose microstructures

K-7174, a GATA-specific inhibitor, is a putative anti-inflammatory agent that attenuates effects of inflammatory cytokines in certain cell types. However, molecular mechanisms involved have not been elucidated. We found that, in glomerular podocytes, induction of monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS) by TNF-alpha was abrogated by K-7174. It was correlated with unexpected induction of unfolded protein response (UPR) evidenced by: (1) induction of endogenous indicators 78 kDa glucose-regulated protein and CCAAT/enhancer-binding protein-homologous protein, and (2) suppression of an exogenous indicator, endoplasmic reticulum stress-repressive alkaline phosphatase. In podocytes, induction of UPR by either tunicamycin, thapsigargin, A23187 or AB5 subtilase cytotoxin completely reproduced the suppressive effect of K-7174. Furthermore, K-7174-elicited UPR abrogated induction of MCP-1 and iNOS not only by TNF-alpha but also by medium conditioned by activated macrophages. These results suggested a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of K-7174.     

Development of a 135K SNP genotyping array for Actinidia arguta and its applications for genetic mapping and QTL analysis in kiwifruit

Kiwifruit (Actinidia spp) is a woody, perennial and deciduous vine. In this genus, there are multiple ploidy levels but the main cultivated cultivars are polyploid. Despite the availability of many genomic resources in kiwifruit, SNP genotyping is still a challenge given these different levels of polyploidy. Recent advances in SNP array technologies have offered a high-throughput genotyping platform for genome-wide DNA polymorphisms. In this study, we developed a high-density SNP genotyping array to facilitate genetic studies and breeding applications in kiwifruit. SNP discovery was performed by genome-wide DNA sequencing of 40 kiwifruit genotypes. The identified SNPs were stringently filtered for sequence quality, predicted conversion performance and distribution over the available Actinidia chinensis genome. A total of 134 729 unique SNPs were put on the array. The array was evaluated by genotyping 400 kiwifruit individuals. We performed a multidimensional scaling analysis to assess the diversity of kiwifruit germplasm, showing that the array was effective to distinguish kiwifruit accessions. Using a tetraploid F1 population, we constructed an integrated linkage map covering 3060.9 cM across 29 linkage groups and performed QTL analysis for the sex locus that has been identified on Linkage Group 3 (LG3) in Actinidia arguta. Finally, our dataset presented evidence of tetrasomic inheritance with partial preferential pairing in A. arguta. In conclusion, we developed and evaluated a 135K SNP genotyping array for kiwifruit. It has the advantage of a comprehensive design that can be an effective tool in genetic studies and breeding applications in this high-value crop.     

Highly sensitive fluorescence and SERS detection of azide through a simple click reaction of 8‐chloroquinoline and phenylacetylene

In 0.19 mol/L acetic acid (HAc), a click reaction of 8‐chloroquinoline/azide/phenylacetylene take places in aqueous solution without Cu(I) as a catalyst. 8‐Chloroquinoline (CQN) exhibited a strong fluorescence peak at 430 nm that was quenched linearly as the concentration of azide increased from 20 to 1000 ng/mL. This quenching was due to consumption of CQN in the click reaction and a decrease in the number of efficiently excited photons due to the presence of triazole–quinoline ramification molecules with strong hydrophobicity. Using blue nanosilver sol as the substrate, CQN absorbed onto the surface of nanosilver particles, showing a strong surface‐enhanced Raman scattering (SERS) peak at 1585 cm^‐1 that decreased linearly as the azide concentration increased from 8 to 500 ng/mL; the detection limit was 4 ng/mL. Thus, two new, simple and sensitive fluorescence and SERS methods have been developed for the determination of azide via the click reaction. Copyright © 2014 John Wiley & Sons, Ltd.     

Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection

The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.     

A new synthesis method for complex electric field patterning using a multichannel dense array system with applications in low-intensity noninvasive neuromodulation

Multichannel coil array systems offer precise spatiotemporal electronic steering and patterning of electric and magnetic fields without the physical movement of coils or magnets. This capability could potentially benefit a wide range of biomagnetic applications such as low-intensity noninvasive neuromodulation or magnetic drug delivery. In this regard, the objective of this work is to develop a unique synthesis method, that enabled by a multichannel dense array system, generates complex current pattern distributions not previously reported in the literature. Simulations and experimental results verify that highly curved or irregular (e.g., zig–zag) patterns at singular and multiple sites can be efficiently formed using this method. The synthesis method is composed of three primary components; a pixel cell (basic unit of pattern formation), a template array (“virtual array”: code that disseminates the coil current weights to the “physical” dense array), and a hexagonal coordinate system. Low-intensity or low-field magnetic stimulation is identified as a potential application that could benefit from this work in the future and as such is used as an example to frame the research.