Building addressable libraries: site selective coumarin synthesis and the "real-time" signaling of antibody-coumarin binding

[reaction: see text] The feasibility of using active semiconductor chips containing addressable arrays of microelectrodes for the "real-time" monitoring of biologically relevant binding events has been demonstrated by detecting the binding of a coumarin substrate by an anticoumarin antibody. The coumarin substrate was synthesized proximal to predetermined electrodes on the chip with the use of a Pd(II) reagent that was itself generated by using the selected electrodes. Once the coumarin was synthesized, its binding to the anticoumarin antibody was detected by monitoring the current associated with a ferrocene-ferrocinium ion redox cycle that was established between the electrodes on the chip and a remote auxiliary electrode.     

Attachment of motile bacterial cells to prealigned holed microarrays

Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual hole on a surface and bacterial cell binding can be controlled by altering the pH, with the greatest occupancy occurring at pH 7.8. Cells attached to hole arrays remained motile for at least 4 h. These data indicate that holed surface structures provide a promising footprint for the attachment of motile bacterial cells to form high-density site-specific functional bacterial microarrays.     

Substrate phage as a tool to identify novel substrate sequences of proteases

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.     

Site-selective chemical etching of silicon using patterned silver catalyst

Si convex arrays and Si hole arrays with ordered periodicities were fabricated by the site-selective chemical etching of a Si substrate using patterned Ag nanoparticles as a catalyst. Ag particles were deposited selectively on the Si substrate by a combination of colloidal crystal templating, hydrophobic treatment and subsequent electroless plating. The obtained Ag patterns were of two different types: network-like honeycomb and isolated-island microarrays. The transfer of ordered patterns fabricated by Ag plating onto the Si substrate could be achieved by the selective chemical etching of a Ag-coated Si area using Ag particles as the etching catalyst. On the basis of this process, it is possible to fabricate negative and positive patterns by changing the arrangement of deposited Ag patterns.     

Aromatic aldehydes as fluorogenic indicators for human aldehyde dehydrogenases and oxidases: substrate and isozyme specificity

Substrate properties of a number of potentially fluorogenic aromatic aldehydes of naphthalenes, phenanthrenes and anthracenes and of some coumarin aldehydes towards various forms of the human and rat aldehyde oxidase and dehydrogenase were examined using absorption and emission spectroscopy. It was demonstrated that recombinant human class 1 aldehyde dehydrogenase (ALDH-1) readily oxidizes naphthalene (except for those ortho-substituted), phenanthrene and coumarin aldehydes, whereas the class 3 enzyme (ALDH-3) from human saliva is active only towards 2-naphthaldehyde derivatives. The observed reaction rates in both cases are comparable to those of the best known substrates, and the Km values are typically in the sub-micromolar range. Aldehyde oxidases (AlOx), which are present in mammalian liver, reveal much broader substrate specificity, oxidizing nearly all the compounds examined, including those of the anthracene series, with maximum activity in the micromolar range of substrate concentration. In rat liver, nearly all AlOx activity was located in the cytosolic fraction.     

In situ controlled growth of well-dispersed gold nanoparticles in TiO2 nanotube arrays as recyclable substrates for surface-enhanced Raman scattering

In this paper, well-aligned Au-decorated TiO(2) nanotube arrays with high surface-enhanced Raman scattering (SERS) enhancement were prepared using a facile in situ reduction and controlled growth approach. The gold nanoparticles are well-dispersed and assembled on the mouth surface and the inside of the TiO(2) nanotubes without clogging. The structure and optical properties of the Au-decorated TiO(2) nanotube arrays have been characterized. The Au-decorated TiO(2) nanotube arrays were employed as SERS-active substrates, which exhibit good performance due to long-range coupling between Au nanoparticles, and TiO(2)-assisted enhanced charge-transfer from Au to Rh6G. The SERS activity of the substrates strongly depends on the crystallite size and level of aggregation. The substrates display significant fluorescence quenching ability and uniform SERS responses throughout the whole surface area. Particularly, good recyclability is shown. The photocatalytic property of Au-decorated TiO(2) nanotube array was exploited to recycle the substrate through UV light photocatalytic purification. The experimental results showed that the substrate is featured by high reproducibility and can be used as a highly efficient SERS substrate for multiple detection of different chemical and biological molecules.     

Application of chemical arrays in screening elastase inhibitors

Protein chip technology provides a new and useful tool for high-throughput screening of drugs because of its high performance and low sample consumption. In order to screen elastase inhibitors on a large scale, we designed a composite microarray integrating enzyme chip containing chemical arrays on glass slides to screen for enzymatic inhibitors. The composite microarray includes an active proteinase film, screened chemical arrays distributed on the film, and substrate microarrays to demonstrate change of color. The detection principle is that elastase hydrolyzes synthetic colorless substrates and turns them into yellow products. Because yellow is difficult to detect, bromochlorophenol blue (BPB) was added into substrate solutions to facilitate the detection process. After the enzyme had catalyzed reactions for 2 h, effects of samples on enzymatic activity could be determined by detecting color change of the spots. When chemical samples inhibited enzymatic activity, substrates were blue instead of yellow products. If the enzyme retained its activity, the yellow color of the products combined with blue of BPB to make the spots green. Chromogenic differences demonstrated whether chemicals inhibited enzymatic activity or not. In this assay, 11,680 compounds were screened, and two valuable chemical hits were identified, which demonstrates that this assay is effective, sensitive and applicable for high-throughput screening (HTS).     

Transferable ordered ni hollow sphere arrays induced by electrodeposition on colloidal monolayer

We report an electrochemical synthesis of two-dimensionally ordered porous Ni arrays based on polystyrene sphere (PS) colloidal monolayer. The morphology can be controlled from bowl-like to hollow sphere-like structure by changing deposition time under a constant current. Importantly, such ordered Ni arrays on a conducting substrate can be transferred integrally to any other desired substrates, especially onto an insulting substrate or curved surface. The magnetic measurements of the two-dimensional hollow sphere array show the coercivity values of 104 Oe for the applied field parallel to the film, and 87 Oe for the applied field perpendicular to the film, which is larger than those of bulk Ni and hollow Ni submicrometer-sized spheres. The formation of hollow sphere arrays is attributed to preferential nucleation on the interstitial sites between PS in the colloidal monolayer and substrate, and growth along PSs' surface. The transferability of the arrays originates from partial contact between the Ni hollow spheres and substrate. Such novel Ni ordered nanostructured arrays with transferability and high magnetic properties should be useful in applications such as data storage, catalysis, and magnetics.     

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.     

Liquid templating for nanoparticle organization into complex patterns

Dewetting of thin films of charged polymer solutions produces complex patterns that can be applied to direct nanoparticle organization on solid substrates. The morphology produced by dewetting can be controlled by the solution properties, temperature, and substrate wetting. In this work, new results on this liquid-template self-assembly system are presented, with special emphasis on producing large arrays of organized nanoparticles. On a hydrophilic substrate with complete wetting, the patterns include polygonal networks and parallel-track arrays that extend over several hundreds of microns. These large structures are formed under well-controlled drying conditions and characterized by scanning electron microscopy, which is better suited for the examination of large as well as small areas than atomic force microscopy. On partial wetting substrates, new patterns are observed, including a complex set of parallel curved bands with variable particle number densities.     

Protein micropatterns using a pH-responsive polymer and light

Protein and peptide microarrays are popular candidates for medical diagnostics because of the possibility for high sensitivity and simultaneous marker screening. To realize the potential of these arrays, new strategies for ligand patterning are needed. We report a method for patterning proteins that utilizes a pH-responsive polymer, deep ultraviolet (DUV) light, and a photoacid generator (PAG). Poly(3,3'-diethoxypropyl methacrylate) (PDEPMA) contains reactive acetal side chains which are converted to aldehydes following treatment with acid. PDEPMA was spin-coated onto Si-SiO(2) substrates and was either chemically deprotected with 1 M HCl or photochemically deprotected by exposure to DUV in the presence of triphenylsulfonium triflate. Conversion to aldehyde groups was confirmed with Purpald and by reaction with a green fluorescent hydroxylamine. Protein microarrays were demonstrated by incubating photochemically patterned surfaces with an aldehyde-reactive biotin followed by red fluorescent streptavidin. This methodology provides a new substrate for the precise patterning of both peptides and proteins for various biological applications including medical sensors.     

Formation and characterization of DNA microarrays at silicon nitride substrates

A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.     

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G?+?C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.     

Growth of high-density parallel arrays of long single-walled carbon nanotubes on quartz substrates

Herein we report a CVD approach to prepare high-density and perfectly aligned arrays of long SWNTs on stable temperature (ST)-cut quartz substrates using copper as catalyst and ethanol as carbon source. Compared with earlier reports, we have demonstrated that the aligned nanotube arrays can be grown on ST quartz substrate without the need of thermal annealing. The density can reach >50 nanotubes per micron and the length can be a few millimeters. Additionally, we have obtained direct proof on the "tip-growth" mechanism for the aligned nanotubes and important evidence that explained the termination of the growth.     

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

Covalent immobilization of carbohydrates on sol-gel-coated microplates

Carbohydrate microarrays have attracted increasing attention in recent years because of their ability to monitor biologically important protein-carbohydrate interactions in a high-throughput manner. Here we have developed an effective approach to immobilizing intact carbohydrates directly on polystyrene microtiter plates coated with amine-functionalized sol-gel monolayers. Lectin binding was monitored by fluorescence spectroscopy using these covalent arrays of carbohydrates that contained six mono- and di-saccharides on the microplates. In addition, binding affinities of lectin to carbohydrates were also quantitatively analyzed by determining IC(50) values of lectin-specific antibody with these arrays. Our results indicate that microplate-based carbohydrate arrays can be efficiently fabricated by covalent immobilization of intact carbohydrates on sol-gel-coated microplates. The microplate-based carbohydrate arrays can be applied for screening of protein-carbohydrate interactions in a high-throughput manner.     

Facile Fabrication of Multi‐targeted and Stable Biochemical SERS Sensors

The direct transfer of single‐crystalline Au nanowires (NWs) onto Au substrates was achieved by a simple attachment and detachment process. In the presence of a lubricant, Au NWs grown vertically on a sapphire substrate were efficiently moved to an Au substrate through van der Waals interactions. We demonstrate that the transferred Au NWs on the Au substrate can act as sensitive, reproducible, and long‐term‐stable surface‐enhanced Raman scattering (SERS) sensors by detecting human α‐thrombin as well as Pb²⁺ and Hg²⁺ ions. These three biochemically and/or environmentally important analytes were successfully detected with high sensitivity and selectivity by Au NW‐SERS sensors bound by a thrombin‐binding aptamer. Furthermore, the as‐prepared sensors remained in working order after being stored under ambient conditions at room temperature for 80 days. Because Au NWs can be routinely transferred onto Au substrates and because the resultant Au NW‐SERS sensors are highly stable and provide with high sensitivity and reproducibility of detection, these sensors hold potential for practical use in biochemical sensing.     

Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

框架核酸高通量检测芯片

构建了一种基于框架核酸的高通量生物检测芯片.利用超微量移液自动化平台,将包含框架核酸探针的液滴按照预设命令固定至生物芯片微阵列上,在探针捕获核酸靶标后利用集成的基因芯片扫描仪对芯片进行成像,通过分析荧光强度定量化分析靶标浓度.结果表明,此框架核酸芯片能够实现框架核酸探针的高通量制备, 24 h即可制备具有15万个点的微阵列,且点间距离的相对偏差W≤10%、荧光强度的变异系数CV=3.30%,具有较高的稳定性,远高于国家标准.此外,该芯片具备高灵敏度、可寻址的高通量生物分析能力,对核酸靶标的检测限可达100 pmol/L.随着多种探针技术的发展,生物检测微阵列技术在高通量生物分析领域展示出巨大的潜力.     

Protein microarrays: prospects and problems

Protein microarrays are potentially powerful tools in biochemistry and molecular biology. Two types of protein microarrays are defined. One, termed a protein function array, will consist of thousands of native proteins immobilized in a defined pattern. Such arrays can be utilized for massively parallel testing of protein function, hence the name. The other type is termed a protein-detecting array. This will consist of large numbers of arrayed protein-binding agents. These arrays will allow for expression profiling to be done at the protein level. In this article, some of the major technological challenges to the development of protein arrays are discussed, along with potential solutions.