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An electrochemical affinity assay system for detection of ligand-ligand receptor binding.
[0001] Affinity assay systems are commonly used in clinical and non-clinical situations to detect, monitor, or confirm the identity, amount, or presence of a particular ligand. Examples include immunoassays for the detection of an antibody or antigen, such as enzyme linked immunoassay (ELISA) or radioimmunoassay (RIA). Such affinity assays confer specificity and sensitivity to the analysis of a particular ligand in a complex sample, such as blood or other body fluid.
[0002] Conventional affinity assays employing conventional labeling and detection techniques typically require washing and/or separation steps. In addition, many affinity assay systems require detection in a machine such as a spectrophotometer or fluorimeter. These are not practical when detection of the ligand in whole blood and other strongly light absorbing or scattering biological fluids is desired. Some conventional affinity assays, not requiring such equipment, rely on visible color changes for detection of ligand, which is also not practical in an opaque or colored fluid such as blood. Furthermore, the commonly used detection compounds, of conventional assays, such as hydrogen peroxide, are rapidly eliminated by protective enzymes found in blood and other tissues, such as catalase.
[0003] An affinity assay providing sensitive, efficient, and rapid detection of a ligand in a complex sample medium, and particularly for detection in whole blood is needed. A preferred assay would not require washing or separation steps, sample removal to machinery for analysis, and most preferably, would utilize only materials contained or generated in its probe, materials available in the biological fluids analyzed or, if added, not rapidly decomposed by enzymes in biological fluids. An affinity assay satisfying these criteria would permit the production of affinity assay systems to detect ligands in whole blood. Such an affinity assay system is described in the instant invention.Summary of Invention
[0004] The affinity assay system of the present invention is based on the electrical connection of the third member, upon its binding to the second member, when the second member is located on or in the redox polymer film on the detecting electrode. The connection of the detection marker to the electrode is via the conducting redox polymer. The affinity assay system of the invention is capable of detecting and/or quantitating a variety of specific ligands, including proteins and nucleic acids, without washing or separation steps. The affinity assay system of the invention operates in whole blood and in other unseparated biological fluids, such as those of tissues and living cell cultures, without added toxic or unstable agents.
[0005] The affinity assay system of the invention includes an electrode coated with a conducting redox polymer, preferably a redox hydrogel. The redox polymer has multiple fast redox centers. The system has at least three members. The first and second members and also the second and third members are capable of conjugating with each other, and therefore capable of binding with each other. The first member of the ligand-ligand receptor pair is immobilized within the redox polymer either through an affinity reaction or by covalent bonding. The second member of the ligand-ligand receptor pair binds to the first. The third member is labeled with an amplifying detection marker, such as a peroxidase. Generation of the detection compound, the substrate of the detection maker, is catalyzed by another enzyme, such as choline oxidase. The enzyme that catalyzes the generation of a detection compound is immobilized in the redox polymer, but its reaction centers are preferably not oxidized by oxidized redox centers of the polymer and are not reduced by reduced redox centers of the polymer when the electrode is poised at its operating potential.
[0006] In a preferred embodiment, the affinity assay system includes an electrode coated with an electron conducting redox polymer in which a strongly binding member of a bioconjugating couple, such as ss-DNA or ss-peptide DNA, avidin, or streptoavidin and a substrate generating enzyme, such as hydrogen peroxide-generating choline oxidase, are immobilized. ("ss" means single-stranded.) The first member of a ligand-ligand receptor pair is biotinylated or labeled with DNA or peptide DNA, and bound to the redox polymer via DNA hybridization or avidin-biotin coupling. The second member is bound then to the first. The third member is labeled with the detection marker horseradish peroxidase or soybean peroxidase. Binding of the labeled third member to the electrode via the second member results in electrical contact between the peroxidase and the redox polymer, causing the electrical connection of the reaction centers of the peroxidase label to the electrode through the conducting redox polymer. Such connection converts the film to a catalyst for the electroreduction of the hydrogen peroxide produced within the film by the immobilized substrate-generating enzyme.Brief Description of Drawings
[0007] Figure1 (A and B) is a pair of schematic diagrams showing the transduction of the concentration of a second member of a ligand-ligand receptor pair, IgG, to a cathodic current.
[0008] Figure 2 is a schematic diagram showing electron transport on binding of the enzyme labeled third member in an immunoassay of the invention.
[0009] Figure 3 is a diagram showing the structure of the redox polymer PAH which forms an electron-conducting hydrogel upon crosslinking.
[0010] Figure 4 is a graph showing the dependence of the current density on the concentration of the second member of the ligand-ligand receptor binding pair, IgG.
[0011] Figure 5 is a graph showing the dependence of the current density on the ratio of the third member, HRP-labeled anti-IgG to the second member, IgG at a fixed concentration of IgG (86 ng/ml).
[0012] Figure 6 is a graph showing the dependence of the current density on the loading of the first member, biotin-labeled anti-rabbit IgG on the redox-polymer film on the electrode at a fixed concentration of IgG (86 ng/ml).
[0013] Figure 7 is a graph showing the dependence of the current density on the concentration of choline, the non-toxic and stable precursor of the detection compound, H2O2.Detailed Description
[0014] DefinitionsAs used herein, the following terms and phrases have the definitions indicated:Redox Hydrogel: The hydrated form of a crosslinked redox polymer.
[0015] Substrate generating enzyme: An enzyme generating or producing the substrate of a detection marker, generally immobilized in the redox hydrogel. The detection marker is usually an enzyme and is covalently bound to the third member. An example of substrate generated by the enzyme is H2O2. H2O2 is generated, for example, by the substrate generating enzyme choline oxidase, which catalyzes the reaction of O2 and choline. Substrate generating enzymes that do not exchange electrons with the redox polymer at the potential where the electrode is poised are preferred. Electron exchange means transfer of electrons from the enzyme to the redox hydrogel or from the redox hydrogel to the enzyme.
[0016] Binding Agent: A macromolecular binding agent of a biomolecule. Examples of binding agents include avidin; streptavidin; single stranded (ss) oligonucleotides; single stranded DNA; and peptide oligonucleotides or peptide DNA. The binding agent is immobilized in or on the redox hydrogel.
[0017] First member of the ligand-ligand receptor pair: A molecule binding to the binding agent and to the second member of the pair; or a molecule bound to the redox hydrogel and binding to the second member. The first and third members are not identical. An example of a first member is a biotinylated antigen.
[0018] Second member of the ligand-ligand receptor pair: Binds to the first and third members. An example of a second member is an antibody or an F(ab′)2 fragment of an antibody against both the antigen of the first member and the antigen of the third member. The electrochemical affinity assay of this invention is usually of the second member.
[0019] Third member labeled with the Detection Marker: Binds to the second member and is labeled with a detection marker, which is preferably a catalyst, such as an enzyme, and most preferably an oxidoreductase. An example of a third member is a peroxidase-labeled antigen.
[0020] Detection Marker: A catalyst, usually an enzyme and preferably an oxidoreductase, labeling the third member. The detection marker can transfer electrons to or accept electrons from the redox hydrogel on the electrode. In these processes the detection marker is electrooxidized or electroreduced. When electrooxidized, the detection marker can oxidize a detection compound. When electroreduced, it can reduce a detection compound.
[0021] Detection Compound: A molecule or ion, or a precursor of a molecule or ion, the electrooxidation or the electroreduction of which produces the detected electrochemical signal, usually a current or potential. The detection compound is electrooxidized or electroreduced in the redox polymer film. An example of an electroreduced detection compound is hydrogen peroxide.
[0022] As shown schematically in Figures 1A and 1B, an affinity assay of the invention includes an electrode 10 coated with a redox polymer 12, preferably a redox hydrogel, in which the first member 14 is immobilized via a binding agent 11 such as avidin. The binding agent 11 is preferably covalently bound to the redox polymer 12.
[0023] In the assay of the invention, a second member 16 of the ligand-ligand receptor pair is conjugated with and thereby bound to the first member 14; then the second member 16 is conjugated with and bound to the third member 20. The third member 20 is labeled with a detection marker 18. In a "sandwich" type immunoassay, the second member is an antibody. It can, however, also be an antigen, if the first and third members are non-identical antibodies to the different regions of the second member antigen. The redox hydrogel 12 further includes at least one substrate-generating enzyme 22, meaning an enzyme generating the detection compound. It is preferred, though not required, that the reaction centers of the substrate generating enzyme 22 not be reduced by the reduced redox centers of the redox polymer, nor oxidized by the oxidized redox centers of the redox polymer, when the redox polymer film 12 is in contact with the electrode 10 and the electrode is poised at its operating potential. The detection compound is, preferably, the substrate of the detection marker 18, which is preferably an oxidoreductase. Binding of the first member 14 to the second member 16, and of the second member 16 to the third member 18, results in an increase in the current passing through the electrode. The increase in current is caused by the catalysis of the electrochemical reduction or oxidation reaction of the detection compound in the combined presence of the detection marker 18 and the redox hydrogel 12 on the electrode. The increase in current correlates with the binding of the second member 16 of the ligand pair, and thereby with the amount of the second member 16 in a sample. This preferred assay system does not require separation or washing steps, permitting use of the assay system in situ in biological fluids. The assay can be carried out in colored and light-scattering media like blood and in media where externally added detection compounds, like hydrogen peroxide, rapidly decompose. In the instant assay hydrogen peroxide is generated in the film on the electrode and is available, at least in part, for electroreduction when the film becomes electrocatalytic through the immobilization of the detection marker, which is bound to the third member.
[0024] Figure 2 shows schematically the flow of electrons in an affinity assay system of the invention. Choline oxidase, a substrate generating enzyme 22, present in the redox polymer 12, generates hydrogen peroxide in the redox hydrogel 12. Binding of a peroxidase-labeled third member 20 to the immobilized second member 16 of a ligand-ligand receptor pair enables peroxidase-catalyzed electroreduction of hydrogen peroxide, a reaction where electrons flow from the electrode 10 to the redox hydrogel 12; from the redox hydrogel 12 to the peroxidase; and from the peroxidase to hydrogen peroxide, the hydrogen peroxide being electroreduced to water.
[0025] The working electrodeThe working electrode 10 is typically a thin film of conductive material disposed on an insulating substrate. Suitable materials for the substrate include, for example, insulating silicon, fused silicon dioxide, silicate glass, alumina, aluminosilicate ceramic, an epoxy, an epoxy composite such as glass fiber reinforced epoxy, polyester, polyimide, polyamide, or polycarbonate.
[0026] A variety of conductive materials can be used to form the working electrode 10. Suitable materials include, for example, carbon, conductive polymers, and metals. Examples of useful metals include gold, platinum, palladium, tantalum, tungsten, and their alloys, as well as metallic compounds like titanium nitride, and ruthenium dioxide. The preferred conductors do not corrode rapidly in aerated aqueous 0.1 M NaCl near neutral pH when a potential of 0.2 volts positive of the potential of the saturated calomel electrode (SCE) is applied. The corrosion current density is preferably less than 10-4A cm-2, and more preferably less than 10-7A cm-2.
[0027] Thin films of these materials can be formed by a variety of methods including, for example, sputtering, reactive sputtering, physical vapor deposition, plasma deposition, chemical vapor deposition, printing, and other coating methods. Discrete conductive elements may be deposited to form each of the working electrodes, for example, using a patterned mask. Alternatively, a continuous conductive film may be applied to the substrate and then the working electrodes can be patterned from the film.
[0028] Patterning techniques for thin films of metal and other materials are well known in the semiconductor art and include photolithographic techniques. An exemplary technique includes depositing the thin film of conductive material and then depositing a layer of a photoresist over the thin film. Typical photoresists are chemicals, often organic compounds, that are altered by exposure to light of a particular wavelength or range of wavelengths. Exposure to light makes the photoresist either more or less susceptible to removal by chemical agents. After the layer of photoresist is applied, the photoresist is exposed to light, or other electromagnetic radiation, through a mask. Alternatively, the photoresist is patterned under a beam of charged particles, such as electrons. The mask may be a positive or negative mask depending on the nature of the photoresist. The mask includes the desired pattern of working electrodes, which are the electrodes on which the electrocatalytic reactions take place when the detection marker and the redox hydrogel are both present and immobilized on the electrode. Once exposed, the portions of the photoresist and the thin film between the working electrodes is selectively removed using, for example, standard etching techniques (dry or wet), to leave the isolated working electrodes of the array.
[0029] The working electrode 10 can have a variety of shapes, including, for example, square, rectangular, circular, ovoid, and the like. The working electrode may have dimension (e.g., length, width, or diameter) which can be 50 µm or less. In some embodiments, the working electrodes are three dimensional structures, and can have a surface area of 1 x 10-4 cm2 or less. Multiple electrodes may be used in an array.
[0030] Counter and reference electrodes may be present in the electrolytic solution off the surface of the substrate containing the working electrode. Alternatively, the counter and reference electrodes may be formed on the substrate containing the working electrode, for example, located on the same or a different surface as the working electrode. It is not necessary for each working electrode to have a dedicated counter electrode or reference electrode. The same counter or reference electrode can serve multiple, or even all, electrodes of an array. A single electrode, such as an Ag/AgCl electrode, can serve as both counter and reference electrode. Preferably the reference electrode is one that does not leach ions and maintains a constant potential. The reference electrode can be, for example, a silver wire or structure, in contact with the electrolytic solution. The surface of the silver wire or structure can be partially oxidized to produce AgCl chemically, or electrochemically. The Redox PolymerThe electrode is coated with a thin film of a redox polymer 12. The redox polymer 12 is deposited on the electrode 10. When multiple working electrodes are used, redox polymer 12 is not deposited on the substrate between the electrodes 10, thus maintaining the electrical isolation of the working electrodes of each other. Another specific method whereby a redox polymer can be deposited, exclusively on the electrode, is electrophoresis. This is a preferred method for coating electrodes of an array when their diameter is small, usually smaller than 50 µm.
[0031] Redox hydrogels are formed of the crosslinked redox polymers upon their immersion in aqueous solutions. The redox hydrogels provide for transport of electrons between the electrode and the detection marker. One type of redox polymer is a redox hydrogel which typically contains at least 10% of water. Water soluble molecules usually permeate through the redox hydrogel rapidly. Electron conduction in the redox hydrogel is believed to occur through electron exchange between polymer segments that, being tethered, do not leach out but are nevertheless mobile within a limited, small radius.
[0032] In general, the redox polymer includes electroreducible and electrooxidizable functions, termed redox centers. These have redox potentials that are a few hundred mV above or below the redox potential of the standard calomel electrode (SCE). Preferably, the redox centers of the potentials of the polymers are not more reducing than about -400 mV and not more oxidizing than about 800 mV versus SCE, and most preferably are not more reducing than about -150 mV and not more oxidizing than about +400 mV versus SCE at neutral pH. The most preferred redox polymers have osmium, ruthenium, or cobalt redox centers and a redox potential ranging from about -150 mV to about +400 mV versus SCE.
[0033] In general, redox polymers suitable for use in the invention have bonds or charges that prevent, or substantially reduce, the diffusional outflux or loss of the redox species during the period of time in which the sample is being analyzed. The bond between the redox species and the polymer may be covalent, coordinative, or ionic. Useful redox polymers and methods for producing them are described in U.S. Patent Nos. 5,264,104; 5,356,786; 5,262,035; 5,320,725; and 5,665,222, incorporated herein by reference. Although many organic or organometallic redox centers can be incorporated in or bound to a polymer and used in the system of the present invention, the preferred redox species are transition metal complexes. The more preferred transition metal complexes are osmium, ruthenium, iron, and cobalt compounds or complexes. The most preferred are osmium and ruthenium complexes.
[0034] One type redox polymer contains a redox species covalently bound to a polymer. An example of such a polymer is poly(vinylferrocene). Another type of redox polymer contains an electrostatically bound redox species. Typically, this type of redox polymer comprises a charged polymer coupled to an oppositely charged redox species. Examples of this type of redox polymer include a negatively charged polymer such as Nafion® (DuPont) in which a positively charged redox species, containing one or more of osmium or ruthenium polypyridyl cations is distributed. Another example of such a polymer is a polymer comprising positively charged functions, such as quaternized poly(4-vinyl pyridine) or poly(1-vinyl imidazole), and negatively charged redox species such as ferricyanide and ferrocyanide. The redox polymer may consist of a highly charged redox species, that itself may be polymeric and have multiple redox centers and an oppositely charged polymer, the redox polymer being bound electrostatically within.
[0035] In another embodiment of the invention, suitable redox polymers include a redox species coordinatively bound to a polymer. For example, the redox species may be formed by complexing of osmium, ruthenium or cobalt ions with 2, 2"-bipyridyl and also with poly(1-vinyl imidazole) or poly(4-vinyl pyridine) or with a copolymer of either of these.
[0036] The preferred redox species are complexes of transition metals, most preferably complexes of osmium, ruthenium, or cobalt. The complexes comprise one or more heterocyclic ligands, each ligand having two or more rings, each ring with one or more nitrogen atoms, such as 2,2"-bipyridine, 1,10-phenanthroline, 2,2',2'', or derivatives thereof. More preferred complexes include osmium cations complexed with two ligands, each ligand containing 2,2"-bipyridine, 1,10-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. In the preferred complexes of osmium, ruthenium, or cobalt, three or more of the coordination sites of the metal ion are nitrogen-occupied, and the number of ligands ranges from 1 to 3. In the most preferred complexes, five of the coordination sites are nitrogen-occupied, and the number of ligands ranges from 2 to 3.
[0037] The preferred redox species exchange electrons rapidly with each other, in a process known as self-exchange and also with the working electrode, so that the complexes can be rapidly electrooxidized and electroreduced. While they can be electrostatically held in the redox polymer, the preferred redox species are coordinatively or covalently bound to the polymer. Those of the preferred polymers that bind the ions of the metal ion complex coordinatively have nitrogen-containing heterocyclic rings, such as pyridine, imidazole, or derivatives thereof. These bind, as ligands, to the cations of the redox species.
[0038] Preferred polymers for complexation with redox species, such as the osmium complexes, described above, include polymers and copolymers of poly(1-vinyl imidazole) (referred to as "PVI"), poly(4-vinyl pyridine) (referred to as "PVP"), and pyridinium-modified poly(acrylic acid). Suitable copolymer substituents of poly(1-vinyl imidazole) include acrylonitrile, acrylamide, acrylhydrazide, and substituted or quaternized N-vinyl imidazole. The osmium complexes coordinatively bind with the imidazole and pyridine groups of the polymer. In copolymers comprising non-coordinating mers, or weakly coordinating ones, such as acrylamide or acrylonitrile, and also strongly coordinating mers such as N-vinyl-imidazole or 4-vinylpyridine, the ratio of osmium complexes to imidazole and/or pyridine groups ranges from 1:10 to 1:1, preferably from 1:2 to 1:1, and more preferably from 3:4 to 1:1. Also, the preferred ratio of the number of complexed transition metal atoms and polymerized vinyl functions ranges from about 1:2 to about 1:30, and more preferably from about 1:5 to about 1:20.
[0039] Examples of other redox species include quinones and species that in their oxidized state have quinoid structures, such as Nile blue and indophenol. The preferred quinones and quinoids do not have hydrogen atoms in their six-membered rings.The redox polymer 12 also preferably includes a binding agent 11 for binding the first member 14 of the ligand-ligand receptor pair. In a preferred embodiment the binding agent 11 is avidin or streptavidin. It binds to the biotinylated first member 14 thereby immobilizing the first member 14 on the electrode.
[0040] The binding agent 11 is preferably covalently bound to the polymer, for example by carbodiimide coupling of carboxylate functions of avidin or streptavidin, to hydrazide functions on the polymer. When the binding agent 11 is an oligonucleotide or DNA, carbodiimide and similar coupling agents activate preferably a terminal phosphate function of these molecules enabling their covalent binding to hydrazide functions of the polymers like PAH of Figure 3.
[0041] Alternatively, the first member 14 can be bound directly, preferably covalently, to the redox polymer by a method such as carbodiimide coupling.
[0042] In one embodiment, the polymer is a copolymer of PVI or PVP with polyacrylamide (it is referred to as "PAA") in which Os(bpy)2 Cl+/2+ is coupled to the imidazole or pyridine functions respectively. To form hydrazides in this redox polymer, for subsequent covalent attachment of the binding agent 11 or for attachment of first member 14, a portion of the amide functions is converted to hydrazide functions by reaction with hydrazine, according to known processes. Typically, at least 5% of the amide groups are converted, preferably, at least 10% of the groups are converted, and more preferably, at least 20% of the groups are converted. The ratio of hydrazide-modified amide groups to unmodified amide groups of the resulting polymer is typically 1:1 to 1:20, and preferably 1:2 to 1:10. The ratio of PVI or PVP to PAA is typically 5:1 to 1:15, preferably, 2:1 to 1:12, and, more preferably, 1:1 to 1:10. The PAA copolymer in which part of the amides is converted to hydrazides is termed PAH.
[0043] In another embodiment, the polymer is a modified poly(acrylic acid). A portion of the carboxylic acid functions of the poly(acrylic acid) are converted to pyridine or imidazole carrying functions. These can be amides, such as those formed with 4-(aminoalkyl)-pyridine, particularly 4-(2-aminoethyl)-pyridine, that can be covalently attached through carbodiimide coupling. The pyridine and imidazole groups can then be used for coordinative binding with the osmium complexes. Typically, at least 2%, preferably, at least 5%, and, more preferably, at least 10%, of the carboxylic acid functions are converted to functions with pyridine or imidazole rings. At least a portion of the remaining carboxylic acid groups are converted to hydrazide groups for crosslinking redox polymer and for covalent attachment of the binding agent or of the first member. Typically, at least 2%, preferably, at least 5%, and, more preferably, at least 10%, of the residual carboxylic acid groups are converted to hydrazide groups.
[0044] A variety of methods may be used to immobilize a redox polymer on an electrode surface. One method is adsorptive immobilization. This method is particularly useful for redox polymers with relatively high molecular weights, for example, greater than about 104 daltons, preferably greater than 105 daltons, and most preferably greater than 106 daltons. The molecular weight of a polymer may be increased, for example, by cross-linking with a di- or polyfunctional cross-linking agent, such as those listed in the Pierce catalog, 1994, pages T155-T167. Examples of functions of cross-linking agents useful in the invention include epoxy, aldehyde, N-hydroxysuccinimide, halogen, imidate, thiol, and quinone functions. Examples of crosslinkers include poly(ethylene glycol) diglycidyl ether and cyanuric chloride, poly(ethylene glycol) diglycidyl ether (PEGDGE) of 400 or 600 daltons being most preferred. Other cross-linking agents may also be used.
[0045] In another embodiment, the redox polymer is immobilized by covalent bonding to a functionalized electrode surface. Carbon surfaces can be modified for covalent attachment of a redox polymer, for example, by electroreduction of a diazonium salt or by oxidation with hydrogen peroxide in the presence of divalent iron ions at a pH of less than 6. As an illustration, reduction of a diazonium salt formed upon diazotization of p-aminobenzoic acid modifies a carbon surface with phenylcarboxylic acid groups. These groups can then be activated by a carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. The activated groups are then bound with a hydrazide or amine-functionalized redox couple, such as, for example, PAH, the polymer of Figure 3.
[0046] The preferred redox hydrogel 12 contains also additional compounds that are useful in the affinity assay of the invention. In particular, it contains the substrate generating enzyme 22. This enzyme catalyzes the generation of the detection compound, which is the substrate of the detection marker 18. The enzyme generates the detection compound by reacting stable and preferably non-toxic reagents. As a result of the internal generation of the detection compound, within the film on the electrode, the need for adding unstable or toxic agents to the analyzed fluid is obviated. This enables the affinity assay of whole blood, for example, where the detection compound, meaning the or substrate of the detection marker of the third member, like hydrogen peroxide, would rapidly decompose.
[0047] An example of a substrate generating enzyme that may be immobilized in the redox polymer is choline oxidase, which generates hydrogen peroxide in situ within the redox polymer film on the electrode. Hydrogen peroxide is catalytically electroreduced when the peroxidase detection marker of the third member is in contact with the redox hydrogel. While in absence of either the peroxidase detection marker or the redox polymer, the film is not electrocatalytic for H2O2 reduction in the applied potential range between 0.1 V (SCE) and + 0.4 V (SCE), the film is electrocatalytic through this range when both the peroxidase and the redox polymer are co-immobilized. The redox or reaction centers of choline oxidase do not rapidly exchange electrons with those of the redox hydrogel, so the presence of the enzyme in the hydrogel and the presence of choline do not change the signal, usually the current or potential. Choline oxidase catalyzes the reaction of molecular oxygen with choline, whereby hydrogen peroxide and betaine aldehyde are formed.
[0048] Ligand-Ligand Receptor PairA ligand-ligand receptor pair, consisting of the first member and the second member, includes any such pair known to bind with specificity. Such ligand-ligand receptor pairs are well known and include the following: antigen-antibody; peptide, e.g. growth factor-receptor; nucleic acid-nucleic acid binding protein; complementary pairs of nucleic acids; and of peptide nucleic acids and nucleic acids.
[0049] When an antibody is used, it is possible to use a fragment, such as an F(ab′)2 fragment. When a nucleic acid sequence is used, an oligonucleotide of less than about 200 nucleotides is preferred.
[0050] The first member 14 of the ligand-ligand receptor pair is immobilized on the electrode 10, by either covalent attachment to the redox polymer 12, and more preferably , through non-covalent affinity binding with binding agent 11.
[0051] The second member 16 of the ligand-ligand receptor pair binds the first member 14. Suitable reaction conditions for such binding are known in the art and vary with the nature of the particular pair used.
[0052] The third member and its detection markersThe third member 20 and the second member 16 are also a ligand receptor-ligand pair. Again, any such pair known to bind with specificity can constitute the pair. Useful detection markers are known, and include catalysts which accelerate the oxidation or reduction, preferably the electrooxidation or electroreduction by the electrooxidized or electroreduced redox polymer, of the detection compound. The acceleration of the electrochemical reaction results in an increase in the current at the electrode, the charge flowing usually via the redox polymer and the catalyst. When a third member 20 is labeled with the detection marker 18 and when it binds to the immobilized second member 16, the current generated at the electrode is increased through electrocatalysis of a reaction of the detection compound.
[0053] The preferred affinity assays of the invention are rapid, specific, efficient, and operate with a high signal to noise ratio, preferably greater than 10. The scheme of an exemplary "sandwich" type immunoassay is shown in Figures 1A and 1B and is demonstrated below in Example 1. It operates with a signal to noise ratio of about 15.
[0054] The preferred affinity assay of the invention can be performed either with no additive to the biological fluid analyzed or with adding only a non-toxic biochemical such as choline. The assay can also be performed in an opaque, colored or light-scattering medium, such as blood; and in the preferred mode it requires no separation or washing steps.
[0055] Preferred Operation and Practice of the AssayIn the preferred mode of operation, the electrode 10 is coated with the crosslinked redox polymer 12 containing the binding agent 11 and the substrate generating enzyme 22, both covalently bound within the redox hydrogel 12. The first member 14 is then bound to the redox polymer 12. This completes the preparation of the electrode for the assay. The electrode is then immersed in the fluid to be assayed for the presence and/or amount and/or concentration of the second member. It is preferred that during this immersion the fluid be moving relative to the electrode, meaning that either the electrode be moving, for example rotating, in the fluid, or that the fluid be flowing when the electrode is stationary. After, or simultaneously with, binding of the second member 16 to the electrode via the affinity reaction with the first member 14, the electrode is exposed to the third member 20. The electrode is poised at a potential where the electrocatalytic reduction or oxidation of the detection compound proceeds, this electrode reaction being catalyzed when the detection marker contacts the redox polymer 12, such contact being made upon the binding of the third member 20 to the second member 16.
[0056] The detection marker of the third memberA variety of catalysts can be used as the detection marker 18. Exemplary catalysts are enzymes that catalyze an electrochemical reaction of a detection compound. A variety of enzymes are useful including, for example, peroxidases for use with hydrogen peroxide, glucose oxidase and glucose dehydrogenase for use with glucose, and lactate oxidase and lactate dehydrogenase for use with lactate. While in the case of hydrogen peroxide the detection compound is catalytically electroreduced and is internally generated in the film, in the case of glucose or lactate, which are abundant in biological fluids like blood, the detection marker is externally supplied. Also, these detection compounds are catalytically electrooxidized when both the glucose oxidase detection marker or the lactate oxidase detection marker is immobilized in or on the redox polymer film and the electrode is poised at a potential between -0.3 V (SCE) and +0.7 V (SCE), preferably - 0.2 V (SCE) and + 0.5 V (SCE) and most preferably - 0.1 V (SCE) and + 0.3 V (SCE). Preferably, thermostable enzymes (enzymes capable of operation for at least 1 hour at 37º C) are used. Soybean peroxidase is one example of a thermostable enzyme.
[0057] The Detection CompoundThe detection compound is a substrate for the detection marker or a precursor of the substrate of the detection member. For example, hydrogen peroxide is a detection compound which is a substrate for the detection marker, peroxidose. ExamplesThe invention may be better understood by reference to the following examples, which are not intended to limit the scope of the invention.
[0058] Example 1Electrochemical ELISAA non-competitive, sandwich type ELISA was started by immobilizing the redox polymer PAH onto a glassy carbon electrode. PAH was made as described in de Lumley-Woodyear et al., 1995, Anal. Chem. 67:1332-1338. The structure of the polymer is shown in Figure 3.
[0059] Standard coatings were formed by mixing 2.5 parts of polymer (5 mg/ml in water); 1.5 parts poly(ethylene glycol 400 diglycidyl ether (PEGDGE) (0.5 mg/ml in water)(technical grade, Polysciences, #08210); 1.0 parts choline oxidase (20 mg/ml in 0.1M NaHCO3) (Sigma #C-5896); and 4 parts avidin (5 mg/ml in 0.1 M NaHCO3) (Sigma #A-9390). A volume of 3 microliters of this solution was applied to the 3 mm diameter glassy carbon electrode. The films were permitted to cure and crosslink for 48 hours in a humid atmosphere and then allowed to dry overnight in air prior to testing.
[0060] The electrodes, containing avidin (binding agent) and choline oxidase (substrate generating enzyme) immobilized within their redox polymer coating, were washed in Dulbecco"s PBS for 15 minutes to rehydrate the polymer hydrogel and to remove any non-covalently bound avidin or choline oxidase. PBS, the pH 7.4 phosphate buffer solution, was made with the sodium (0.008 M) and potassium (0.002 M) phosphate and with sodium (0.14 M) and potassium (0.01 M) chlorides. The rehydrated electrodes were then soaked in 5 ml PBS containing 9 micrograms of biotin-labeled anti-rabbit IgG (first member of the ligand-ligand receptor pair) for 30 minutes, followed by washing 10 minutes in PBS to remove any unbound biotin-anti-IgG. This completed the preparation of the electrode for the assay of rabbit IgG.
[0061] The completed electrodes were then placed in a test cell containing 17.5 ml of 20 mM choline in PBS and a potential of -70 mV vs SCE was applied. The electrodes were rotated at 1000 rpm. A sample amount of IgG (second member of the ligand-ligand receptor pair) was added, and the mixture permitted to incubate for 30 minutes. At the end of the incubation period, 6.0 micrograms of HRP-labeled anti-rabbit IgG (third member, labeled with detection marker) was added and the resulting increase in reduction current was monitored. The currents were recorded 30 minutes after the addition of the HRP-labeled probe.
[0062] Figure 4 shows the dependence of the hydrogen peroxide electroreduction current on the concentration of the ligand, rabbit IgG. The current increased linearly, with the rabbit IgG concentration over the 1-1000 ng/ml range. A linear regression analysis of the data yielded an R2 value of 0.89 and a negative current at the intercept where the rabbit IgG concentration was nil, suggesting that some rabbit IgG was lost from the solution through adsorption on the untreated wall of the Pyrex glass test cell.
[0063] To assess the dependence of the signal-to-noise ratio on the concentration of the HRP-labeled anti-rabbit IgG probe, the dependence on the current density on the molar ratio of HRP labeled anti-rabbit IgG probe to the ligand IgG was determined. As shown in Figure 5, in the absence of ligand IgG there was very little noise associated with non-specific binding of the HRP-labeled anti-rabbit IgG probe. At a 4:1 ratio, the signal (specific binding) to noise (non-specific binding) ratio was 15. The results of the ensemble of control experiments, in which individual components of the test system were deleted, are shown below in Table 1. 1 TABLE 1 Electrode Constituents Antigen Antibodies PEGDGE Choline Rabbit Biotin: HRP: Reagents Signal Polymer 400 Oxidase Avidin lgG Ab Ab Choline &mgr;A/cm2 + + − + + + + + 0 + + + − + + + + 0.12 + + + + − + + + 0-0.1 + + + + + − + + 0.17 + + + + − + − + 0.03 + + + + − − − + 0.03 − − − − + − + − 0 − − − − − − − + 0 + + + + + + + + 1.03 + + + + + + + + 1.06
[0065] The residual current density, following deletion of any of the essential components, was equal to or smaller than the current density when the analyte itself (rabbit IgG) was absent. In the presence of choline, but without any immunoreagent, the current density was only 0.03 µA/cm2, showing, as expected, that choline oxidase was so poorly "wired", if at all, that electroreduction of choline did not interfere with the assay. When the avidin-containing redox hydrogel was not activated with biotin-labeled anti-rabbit IgG, the current density was 0.17 µA/cm2, about one fifth of that observed in a typical assay.
[0066] The dependence of the current density on the biotin-labeled anti-rabbit IgG loading of the electrodes is shown in Figure 6.
[0067] In a separationless assay when the concentration of the rabbit IgG, F(ab′)2 fragments was 86 mg/mL and the concentration of the HRP-labeled anti-rabbit IgG was 344 mg/mL. (HRP is horseradish peroxidase), and with only choline added to the test solution, a current density of 1.5 µA cm-2 was observed.
[0068] Varying the ionic strength of the test buffer influenced the level of both the signal and the noise. Both signal and noise decreased rapidly as salt content increased, with the noise level dropping to essentially zero for salt concentrations of 0.2 M and above.
[0069] When the F(ab")2 antibody fragments were replaced with whole antibodies, the signal to noise ratio was about 4:1 under identical conditions.
[0070] Study of the effect of the duration of the steps of (a) the activation with biotin-labeled anti-rabbit IgG, (b) the binding of IgG and (c) the binding of HRP-labeled anti-rabbit IgG were examined when the concentrations of the three reagents were, respectively, 1800 ng/ml, 86 ng/ml, and 344 ng/ml. The signal current reached its plateau after 15 minutes of incubation in the activation step. When the incubation time was halved to 7.5 minutes, 75 % of the current was retained. In the rabbit IgG exposure step, the current plateau was reached in 15 minutes, and shortening of the incubation period to 10 minutes resulted in only a 10% reduction in current. For the final step of binding HRP labeled anti-IgG, the current did not level off in 30 minutes, but increased linearly with time, becoming easily measurable in about 5 minutes.
[0071] These data demonstrated a separationless electrochemical immunoassay system, requiring no washing steps, nor toxic or unstable additives to the analyzed fluid. The platform of the assay is a redox hydrogel that conducts electrons, in which avidin and choline oxidase are co-immobilized. This hydrogel is permeable to large biological molecules, including antibodies and enzyme labeled antibodies. The choline oxidase in the gel catalyzes the reaction of choline and oxygen, whereby betaine aldehyde and hydrogen peroxide are produced within the sensing layer. Hydrogen peroxide is electroreduced in this layer to water at potentials negative of 400 mV (SCE) preferably between -70 mV (SCE) +150 mV (SCE), after components of the electrocatalyst are assembled in the sandwich of the immunoassay. Once the platform of co-immobilized avidin and choline oxidase in the electron conducting hydrogel is in place, the probe is activated with the desired biotinylated immunoreagent. If the assayed fluid contains the complementary immunoreagent and if a peroxidase-tagged probe of the immunoreagent is added, a cathodic current flows as a result of the catalyzed electroreduction of the H2O2. The detection limit of the assay is about 4 ng/ml for the IgG/anti-IgG system, and the assay of a sample is completed within about 20 minutes or less.
[0072] The magnitude of the observed current densities, 2 µA/cm2, was high enough to provide for miniaturization to 10 micrometer diameter electrodes, sufficient to assure that mass transport and electron transport both be radial. Because in microelectrodes, where electron transport between the "wired" enzyme and the electrode is radial, the current density is higher by a factor of 4π than in the semi-infinite planar electrodes of the Example, it is estimated that the current will be of about 20 picoamperes in the 10 micrometer diameter electrodes.
[0073] The present invention should not be considered limited to the particular examples described above, but rather should be understood to cover all aspects of the invention as fairly set out in the attached claims. Various modifications, equivalent processes, as well as numerous structures to which the present invention may be applicable will be readily apparent to those of skill in the art to which the present invention is directed upon review of the instant specification.
1. 18.A method for detecting a second member of a ligand-ligand receptor pair in a sample; the method comprising:
- contacting the sample with an electrode having immobilized thereon a first member of the ligand-ligand receptor pair and with a third member that binds the second member to form a first member-second member-third member complex, the third member labeled with a detection marker;
- wherein the detection marker-labeled first member-second member-third member complex generates an electrical signal when a potential is applied to the electrode; and
- correlating the generated electrical signal with the presence or amount of the second member in the sample.
2. 19.The method according to claim 18, wherein the first member is immobilized in a redox polymer on the electrode.
3. 20.The method according to claim 18, wherein the electrode further comprises an immobilized substrate generating enzyme, the substrate generating enzyme generating a detection compound.
4. 21. The method according to claim 20, wherein the substrate generating enzyme is immobilized in the redox polymer.
5. 22.The method according to claim 20, wherein the substrate generating enzyme comprises one or more reaction centers, and wherein the reaction centers do not exchange electrons with the electrode.
6. 23.The method according to claim 21, wherein the substrate generating enzyme comprises one or more reaction centers, and wherein the reaction centers do not exchange electrons with the redox polymer.
7. 24.The method according to claim 20, wherein the substrate generating enzyme comprises choline oxidase.
8. 25.The method according to claim 21, wherein the substrate generating enzyme comprises choline oxidase.
9. 26.The method according to claim 18, wherein the first member is immobilized in a film coating the electrode.
10. 27. The method according to claim 26, wherein the film comprises a binding agent that binds the first member.
11. 28.The method according to claim 18, wherein the first member is non-covalently bound to a film coating the electrode.
12. 29. The method according to claim 27, wherein the binding agent covalently binds the first member.
13. 30.The method according to claim 27, wherein the binding agent is avidin or streptavidin, and the first member is biotinylated.
14. 31.The method according to claim 20, wherein the detection compound comprises hydrogen peroxide.
15. 32.The method according to claim 18, wherein the detection marker comprises peroxidase.
16. 33.The method according to claim 32, wherein the peroxidase is horseradish peroxidase or soybean peroxidase.
17. 34.The method according to claim 18, wherein the potential applied to the electrode is not more reducing than -0.0V versus the saturated calomel electrode (SCE) and not more oxidizing than +0.4V versus the saturated calomel electrode (SCE).
18. 35.The method according to claim 18, wherein contacting further comprises the step of rotating the electrode within the sample.
19. 36.The method according to claim 18, wherein the sample comprises blood.
20. 37.A method for detecting a second member of a ligand-ligand receptor pair in a sample; the method comprising:
- contacting the sample with a sensor wherein the sensor comprises;
- an electrode;
- a redox polymer film on the electrode;
- a substrate generating enzyme on the electrode that generates a detection compound;
- a first member of the ligand-ligand receptor pair that binds the second member, the first member on the electrode;
- and with a third member that binds the second member, the third member labeled with a detection marker that reacts with the detection compound to form a first member-second member-third member complex;
- wherein the detection marker-labeled first member-second member-third member complex generates an electrical signal in the presence of the detection compound when a potential is applied to the electrode; and
- correlating the generated electrical signal with the presence or amount of the second member in the sample.
21. 38.The method according to claim 37, wherein the substrate generating enzyme comprises one or more reaction centers, and wherein the reaction centers do not exchange electrons with the redox polymer.
22. 39.The method according to claim 37, wherein the substrate generating enzyme is choline oxidase.
23. 40.The method according to claim 37, wherein the redox polymer film on the electrode further comprises a binding agent that binds the first member.
24. 41.The method according to claim 40, wherein the binding agent covalently binds the first member.
25. 42.The method according to claim 40, wherein the binding agent non-covalently binds the first member.
26. 43.The method according to claim 40, wherein the binding agent is avidin or streptavidin, and the first member is biotinylated.
27. 44.The method according to claim 37, wherein the detection compound is hydrogen peroxide.
28. 45.The method according to claim 44, wherein the detection marker is peroxidase
- 46.The method according to claim 45, wherein the peroxidase is horseradish peroxidase or soybean peroxidase.
29. 47.The method according to claim 37, wherein potential applied to the electrode is not more reducing than -0.0V versus the saturated calomel electrode (SCE) and not more oxidizing than +0.4V versus the saturated calomel electrode (SCE).
30. 48.The method according to claim 37, wherein the contacting further comprises rotating the electrode within the sample.
31. 49.The method according to claim 37, wherein the sample comprises blood.
32. 50.The method according to claim 37, wherein the first member of the ligand-ligand receptor pair is an antigen and the second member of the ligand-ligand receptor pair is an antibody.
33. 51.The method according to claim 37 wherein the first member of the ligand-ligand receptor pair is a growth factor receptor and the second member of the ligand-ligand receptor pair is a growth factor.
34. 52.The method according to claim 37 wherein the first member of the ligand-ligand receptor pair and the second member of the ligand-ligand receptor pair are complementary strands of nucleic acid.
Type: Application
Filed: Jun 18, 2001
Publication Date: Sep 26, 2002
Patent Grant number: 6576461
Inventors: Adam Heller ( Austin , TX ), Charles N. Campbell ( Austin , TX )
Application Number: 09883591
International Classification: G01N033/53; C12M001/34;


text:
The Scottish Terrier, CH Roundtown Mercedes Of Maryscot, has to be one of the most recognizable show dogs in recent history. A Google search of "Sadie, the Scottish Terrier" uncovered 375 news articles in the past week! Not 375 references in obscure show dog rags like this humble blog, but in the mainstream media like USA Today, the Boston Herald, and the New York Daily News. Sadie is, of course, 2009's number one dog all breeds, winner of 111 career all breed Bests, and winner of the 2009 AKC/Eukanuba National Championship, and, according to Las Vegas odds makers, the 1:8 favorite to win Westminster. Sadie was bred by Mary O'Neal & Anstamm Kennels (Miriam "Buffy" Stamm and Cindy Cooke), is owned by Amelia Musser, and handled by Gabriel Rangel.
Sadie has made only infrequent appearances in the show ring during 2010, but our current number one Terrier, the Smooth Fox Terrier, CH J'Cobe Kemosabe Vigilante Justice, has been pillaging the show rings, garnering eight BIS so far this year, more than any other dog out there. Dodger and handler Amy Booth finished 2009 in the number five spot all breeds and are currently a breath behind the number one dog. Dodger was bred by William Dalling & Stacy Turner, and is owned by Sandra & Howard Hoffen and Phil & Amy Booth. Amy does most of the handling, but occasionally Phil steps in as he did in yesterday's BIS performance in Indianapolis.
Scott Sommer, handler of last year's Westminster winner, the Sussex Spaniel, CH Clussexx Three D Grinchy Glee, will have several entries this year including the Wire Fox Terrier, CH Dalriada's Mystic. Although Boo joined the fun late in 2009, she still managed five BIS, the only Wire Fox to win an all breed Best. Breeder Cara Campbell DVM co-owns Boo with Mike Doleski, Cecilia Ruggles & Beth Dowd.
The Skye Terrier, CH Cragsmoor Buddy Goodman, 2009 Westminster Breed winner and the only Skye to win a BIS in 2009 (He won seven), is currently the country's number two Terrier and has just come off a very impressive weekend in Atlanta. Buddy was bred by Eugene Z Zaphiris & Matthew H Stander, is owned by Carolyn Koch & Victor Malzoni Jr, and handled by Lawrence W Cornelius.
Stay tuned for the Toys.

subset: commoncrawl


text:
Top 10 Motorcycle Memes
Don't Miss Stories on Motorcycle.com
1. The NFL's Deflated Balls
The meme that inspired this Top 10 list. The irony of this joke, however, is that, while motorcycle racers undoubtedly have bigger stones than the rest of humanity, what makes them great – beyond their obvious natural talent – is their thoughtfulness. Anyone who has had the privilege of spending time observing racers as they go about their job will tell you their singular focus and relentless desire to do whatever it takes to win is a sight to behold. Of course, I would be remiss if I didn't mention that the incredible creativity a racer's mechanics exhibit in their interpretation of the rules is another thing that makes motorcycle racing great.


text:
vegan pizza
When I went vegan at 17 my Italian mother was not happy at all. Mostly she feared I'd starve to death without manicotti, ravioli, lasagna or pizza. In the mid-90's vegan cheese consisted of only tofutti slices that are about as similar to cheese as the package they come in. But I was motivated by the likes of Chokehold and Earth Crisis (though maybe not enough to have Firestorm karaoke at my wedding) and was committed to never eating these foods again.
homemade eggplant pizza as seen on Pizza Intolleranza
My how times have changed. 'Which vegan cheese do you use?' is now a normal question at pizza places. Just got back from a short trip to NYC and ate more 'cheese' pizza than I have in years. Pretty stoked though I hope people still work on developing great recipes that aren't dependent on vegan cheese. On some levels that is happening as is evident by my friend Gian's vegan pizza blog. I recently sampled some and was quite pleased. Since I developed my lasagna de bartolomeo vanzetti recipe (as seen in Please Don't Feed the Bears) I have not played around with Italian recipes nearly enough. Will do though. Thanks for motivation, Gian.


text:
- 225g crunchy peanut butter
Peanut butter
pee-nut butt-ah
Peanut butter starts with dry roasting peanuts, which concentrates and heightens their flavour…
- 200g bar dark chocolate, broken into pieces
Dark chocolate
dahk chok-lit
Dark chocolate means the shiny, dark-reddish brown treat produced from the cacao bean, theobroma…
- 280g soft light brown sugar
- 3 medium eggs
- 100g self-raising flour
Set aside 50g each of the peanut butter and chocolate. Heat oven to 180C/160C fan/gas 4 and line a 20cm square baking tin with baking parchment. Gently melt remaining peanut butter, chocolate and all the sugar in a pan, stirring occasionally, until the sugar has just about melted. Turn off heat and use a wooden spoon to beat in the eggs one by one. Stir in the flour and scrape into the tin.
Melt reserved peanut butter in the microwave on High for 45 secs, or in a pan, until runny, then drizzle over the brownie. Bake for 30-35 mins until it has a crust, but the middle still seems slightly uncooked.
Melt reserved chocolate, drizzle over the brownie, then cool in the tin before cutting into squares.


text:
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Decals 101
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Fine Clonier [MOD]
Joined: 05 Feb 2005
Location: In the Lab, Texas
Posted: Thu Aug 11, 2005 11:45 am
Post subject: Decals 101
This is a cut and paste document, so please excuse the flow, but all the information is in there. Please refrain from posting in this thread unless it is a HOW TO Technique and you have done it quite a bit. If you have questions please start a new thread:
Decals 101 - Questions
What do you need to know?
Ok, the easy question is to hang out or read the forum on FBTB or my forums:
Minifig Customization Network
So summarize the high points of these two great resources I have cut and pastes parts of different threads below, if it doesn't follow exactly this is why.
To remove the current design from your figure you need a micro abrasive: Brasso, which is carried at most major walmart type stores.
I try not to use paint unless there is no other way, then the airbrush is king, however a cheap and nice alternative is Sharpie brand markers, but both Testors and Citadel paints work well and is seems to be completely personal preference between them. I use water slide decals that I print out with a specialized printer, however you can do the same thing with your inkjet. My printer prints in white, silver, and gold ink so it is a bit different. The only place that I know of to get waterslide paper, Decal Paper, is either a hobby store or online. I recommend
If you don't need much get a sample pack, or you can try
and they have a sample pack as well.
Either you use a special printer like mine or you use white decal film. Yes they make it in white, but this requires you to print the torso color in the background areas and reguires really close color matches. check here for color matches:
Color Guide
These are close but not always exact for all printers and requires a bit of playing with the values.
Here is a minifig template in jpg format:
Creation and application of waterslide decals
Jared "Kaminoan" Burks
There are two types of graphics programs, raster and vector. Raster pictures are made of of tiny squares of color. Gif and Jpg the two main graphic formats of the web are raster image formats. When you zoom in, these images become blocky and pixilated. Vector formats are all mathematical. EPS, to some extent PNG and native formats like AI (Adobe Illustrator) or CDR (Corel Draw) are vector formats. When you zoom in on these, they stay sharp and clean because the same math applies at whatever the magnification. Vector graphics are used in illustration and design (Commercial Artwork), most home users, doing simple web graphics, drawing pictures, or photo editing don't have a need for them. However for the best results it is important that you create your designs in a vector art program. An option you might consider is trial version of the aforementioned programs, or Paint Shop Pro.
After selection of your art program and familiarization with that program you can begin creating designs with templates for the torso and legs found here: Minifig Blank:
When creating art for use in the creation of decals it is important to remember the limitation of your printer. Most printers have difficulty in printing a line with a weight of less than 0.3 points (Line weight refers to line thickness). The point size I commonly use in 0.5-0.7. If I need a highlight or a really fine fold or something then I will go down to 0.4 and rarely to 0.3. I do this for several reasons: 1, I draw designs for the FBTB group and want them to have good results when printing (Various printer/color copier types); 2, I convert to jpg format (a lower resolution format for compression into a smaller file size for easy up and download from the net), this means I lose some details, and thus my details need to be bigger; and 3, I try to stay in a similar design scheme as the Lego artist.
Draw and color your design as you would like, remember you can use Legos color palette for their brick colors, which can be found:
Official Lego Brick Color Values (Found on Lugnut)
And an independent assessment of the Lego palette can be found here:
Britdog Models' Lego Brick Color Values (Goggle Search)
How you color your design depends on the media that you are going to print. Options include waterslide decal paper, overhead transparencies, clear self-adhesive stickers, and paper. If you are printing on a clear media keep in mind that the torso color is going to show through and contribute to you design.
Before printing on any expensive media always print a test page. Print your designs out on a piece of scrap material or paper. Confirm your color choices, colors on your screen will not match exactly to printed colors. Confirm your details, you have been working on something very small and in a vector based program you can draw detail much too fine to print.
My specialty is waterslide decals; therefore I am going to discuss this technique. I print using a very specialized micro-dry printer; however, as most people have access to inkjet printers or color copies (Kinkos, etc.) I am going to give instruction on creation of decals using these types of devices. You will need to purchase waterslide label paper from some vender; I use Micromark (www.micromark.com). If using an inkjet printer you need to order media specific for that device, however if you are printing your designs on a color copier, you will need waterslide paper for a laser printer. Print your designs out with the highest resolution possible for your printer (just like you did when performing your test print). Once printed be very careful not to handle the sheet of decals until the ink dries. After you have printed using an inkjet or color copier, you must overcoat the decals with clear spray paint, available at any home improvement store. Apply several thin coats and allow them to dry between applications. This will protect the ink from the water used in the decals application, even if you have waterproof ink in your inkjet printer, this is critical. Once printed cut the decals from the page and follow the manufactures instructions for application of your decals.
There are chemical kits that aid in the application of these types of decals. These kits help make the decal disappear when applied properly, thus making the design appear painted on rather than decal applied. They can be found at many hobby stores, I purchased mine from Micromark (www.micromark.com), however kit components can be found at Internet Trains (www.internettrains.com/bamopa.html). Follow the instructions that accompany the decal application kit for their use and check Fred Lists and Jim Bakers sites for tricks with these kits.
Check the instruction sections on my website:
or RB website. I have found using a q-tip instead of a paint brush works better. I have step by step picture instructions on the site.
As for chemicals this is actually pretty accurate:
You can get a nice application without them (The q-tip really helps), and if you are only making one or two customs, I would skip them. If you are planing on making this a hobby, I would get the decal setting, decal softening, and satin from this kit. They can all be ordered independently. I have yet to use up my first kit.
However you only really need them when applying decals to Legs or arms. The torso and head it doesn't make that big a difference. I do recommend strongly the use of a clear sealent over the top of the decal. Clear gloss hobby paint works well.
Here is a part of Red Bean's site where he compares old fashion stickers to waterslide:
However on re-reading this thread, I don't think that the author is looking for overcoat solutions, I think you need decal setting and decal softening solutions. Check this out:
These are the solutions used to make a decal look painted on instead of applied. However some of the same results are obtainable with good application technique. If you use a q-tip when applying decals that is moist (Not soaking wet) and roll over the top of the decal to remove all bubbles and the excess water, very nice results can be achieved. Now this will not work in all cases, for example R2 domes or Clone Helmets. These complex curves require the softening solution to really look nice. Oh and using the setting solution really reduces decal damaged when the decals are applied to the legs.
Any other questions?
As for overcoats there are many more including nail polish (yellows in sunlight, don't recommend), Future's floor wax (sworn by many model makers for protecting decals), Spray paint (messy), Airbrush (slightly better than spray paint), then liquid overcoats (I like Badger and apply in a similar manner to what Recluse Mage suggests, only I find a hair drier helps the process, helps give a smoother finish and removes any air bubbles that occur. The only draw back is you have to have any excess overcoat removed before you hit it with the hair drier.)
All the pricing and everything was in the link I provided, here it is again:
Well the set of 6 solutions in the kit is $15.40 + Shipping
All I actually use from the kit is:
Decal Setting Solution $2.40
Decal Softening Solution $2.40
Clear Satin $3.10 (I prefer it to the gloss)
$7.90 Total + Shipping (From Micromark there are other vendors and the price is always about the same. They can't ship this out of the US so if you are elsewhere in the world you will have to find a domestic vendor.)
I bought the kit and I have yet to use the whole thing and trust me, I have made a ton of figures.
Original Post:
As far as sculpting I would refer you to Robert Tothiro Martin or Red Bean at MCN they are the best I have seen. To summarize the high points Super Sculpey III works well. Boil or hair drier it don't bake. It can be found at most hobby stores or even Walmart. For more information visit RB's site:
Decal sizes:
1.422 cm Wide
1.201 cm Tall
1.4 cm Wide
1.1 cm Tall
1.4 cm Wide
0.2 cm Tall
To remove the current design from your figure you need a micro abrasive: Brasso, which is carried at most major walmart type stores (Walmart, Target, Grocery stores, etc.).
Paper Towel
Step 1 - Put brass on paper towel.
Step 2 - rub minifig part vigorously against brass-ed paper towel. Apply more brasso if necessary.
Step 3 - Once original print is removed, was piece in soapy water and dry.
Jared "Kaminoan" Burks
Kaminoan's Fine Clonier Decals
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text: Salvation Prophecy is a space epic. Four warring factions battle over control of the known galaxy. Space stations and planet colonies endure constant assault. You begin your journey as a soldier recruit. Through intense planet and space battle, your combat skills will be put to a grim test. Success is rewarded with powerful skills, combat gear, and ship upgrades, and you may rise in rank to become commander of your faction. Lurking in the dark recesses of the universe is a threat foreseen by an apocalyptic prophecy. A danger far greater than mankind's endless battles. The pursuit of the prophecy leads through distant alien worlds with deadly creatures and untold secrets. A chance for redemption, if only we find our way through that dark hour.


text:
Steve MacIntyre is one of those guys who's always up and down from the NHL, and garners more penalty minutes than scoring chances. So, even with the Wilkes-Barre/Scranton Penguins, he's exactly the sort you'd expect to completely lose his mind and challenge the entire opposing team to a fight. All at once? No problem. Wearing goalie pads? Just more of you to punch.
Volume up for this, as the announcers are great.


text:
Mata makes title omission
Juan Mata admits he and his Chelsea teammates have been left disappointed by yesterday's Boxing Day draw with west London rivals Fulham at Stamford Bridge.
After a tightly contested first-half, the Blues broke the deadlock when the Spanish playmaker netted his fifth goal of the season just two minutes after the interval, but Andre Villas-Boas' side were pulled back by a Clint Dempsey strike and the game finished 1-1.
"My goal wasn't enough and we are very disappointed with the point because we needed three points," said Mata. "We had so many chances to score.
"Particularly in the last 15 minutes - with Fernando [Torres], with Didier [Drogba] and with Raul [Meireles], but it wasn't enough. For sure, their goalkeeper made some very good saves so congratulations to him.
"We tried to move the ball quickly but it is difficult when the other team has a lot of players behind the ball and it was difficult for us to find the spaces."
Chelsea now face the prospect of dropping into fifth place in the Barclays Premier League table if Arsenal beat Wolves at the Emirates Stadium this afternoon, and Mata concedes that the club's title aspirations have suffered another setback following yesterday's result.
"It is going to be very difficult because there are lot of teams and they are having very good seasons like Arsenal, like Tottenham, like Liverpool so we have to fight," he added.
"Our next game is another that we have to win but Aston Villa is a very good team. We wanted to achieve the six points in our two home games but against Fulham we got one, and we hope to win three points against Aston Villa."News Now - Sport News


text:
I'm debating on whether or not I want to keep this comic. At least, keep it here. I've been having serious thoughts about placing it on my own site to deal with at my leasure. . .
How do I kill a comic?
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text:
By Ed Harris
Southington Community Cultural Arts (SCCA) is well into its fundraising efforts to turn the old Gura Building into an arts hub, thanks to $500,000 from the State Bond Commission.
"I want to thank former State Representative Zeke Zalaski and current State Representative Dave Zoni for their passionate advocacy in support of the project," said State Rep. Joe Aresimowicz in a release on the bonding decision. "I also want to thank Dawn Miceli and Mary DeCroce for getting it off the ground and moving it forward. The arts center will be wonderful for the Southington community—it will give young people the opportunity to discover and improve their talents."
Southington Town Councilor Dawn Miceli, who has been at the forefront of the effort, said the grant process was two years in the making and was double what SCCA was originally asking for.
Last month, Mary DeCroce, chairperson of the SCCA, updated the Town Council on other funding that the arts group is applying for.
"We've been very busy," said DeCroce.
The group is in the process of applying for three large grants. If approved, the grants would bring $325,000 through a DECD Historic Tax Credit, a $200,000 restoration fund grant (used as reimbursement) and $257,000 in the form of state bonds.
In total, the SCCA has 17 grant applications in various stages of submission, DeCroce said.
Personal pledges have raised $176,000 and numerous fundraisers are planned for the coming months, DeCroce added.
The Gura Building used to house several town offices, but the building was vacated as the new Municipal Center opened.
After a couple litigious Town Council meetings, SCCA was allowed to lease the building for one dollar a year and begin an 18-month fundraising period to raise 80 percent of the renovation costs. This would require SCCA to raise $1.36 million.
The group was optimistic from the get go, bolstered by agencies like the Connecticut Trust for Historic Preservation, which funded a feasibility study on the project, classifying the Gura Building as historically valuable.
To go along with hosting events and exhibiting artwork, the proposed arts building would also include a number of services, including an art supply store and studio space rental.


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Cactus Bistro: First Look
This is not a review of Cactus Bistro, just a first look at a long-anticipated restaurant (Cactus Bistro was originally slated to open Winter 2011). For a review, I prefer to wait at least a month after a restaurant's opening to let a restaurant settle into itself—new staff, new suppliers, new everything tends to result in an inaccurate reflection of what the restaurant experience will ultimately be. (Though a friend also believes in a "six-month suck cycle" in which after the luster fades, the staff begins to slack off. But that's a whole other debate.)
John Memering, previously the executive chef of Kalapawai Cafe, has opened Cactus Bistro right across the street. The first official day of service was yesterday, starting with lunch only, with dinner service to start in May.
Billed as "a celebration of the cuisines of the new Americas with Aloha," the menu channels Latin American flavors. A sample of menu items: Argentine fry bread, Big Island boar picadillo empanadas, feijoada, lomo saltado (a Peruvian stir-fry), and a variety of tacos with fillings from turkey chorizo to pork al pastor. For dinner, the menu transitions away from tacos and tortas (sandwiches) to bigger plates such as duck "carnitas," slow-roasted pork shoulder, and clams with lemon-chile fideo (noodles).
Lunch prices range from $4 to $15, dinner entrees from $15 to $24.
767 Kailua Rd., 261-1000, cactusbistro.com


text: Inspired by the formality and pageantry of imperial design motifs, Campaign is a cast and extruded aluminum collection featuring sling and woven seating options. Handsome details include a cast finial on the arms, cast buns around each joint and at the feet, and epaulets grounding the cross-braces. The woven seat and bucket is fully "upholstered" (wrapped front and back) - customary for all Brown Jordan woven product.


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Product code:
Minimum and Maximum Door Frame Sizes (mm)
Minimum Frame Width: 836 mm
Maximum Frame Width: 1015 mm
We can provide 15mm, 25mm or 50mm frame extensions, side panels and top lights if your sizes are larger than stated.
2 Panel 2 Square 1 Arch Crystal Tulip Composite Front Door in Blue
Made to measure 2 Panel 2 Square 1 Arch Crystal Tulip Composite Front Door in Blue. This new replacement composite front door, pre hung in the frame can be delivered within 5-10 working days from time of order, ready to install.
Overlay Art is decorative glazing at its peak. A large range of brilliant cut bevels is first bonded to the glass, and then painstakingly outlined by hand with highly skilled leadwork. With this design on the external face, natural weathering will tarnish and add to the depth of colour in the lead, whilst the hand cut bevels will retain their beautiful sparkle, giving a stunning yet traditional finish for years to come.Now £639 inc VAT Supply Only
Was £739- £100 Off
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Design, price and order your door through the design tool below or call 01530 273365 or low call rate 0844 692 3828


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Dear Gentleman,
Some days Ago when I was working my Lenovo Idea Pad U350 laptops that time I will downloaded from the lenovo server Bios Update file and next I will trying to install this file but in the mean time my laptop was hanged and several times i will trying to normally shutdown but it does not shutdown on normally?
At that situation I was waiting for the some times regarding for normal shutdown? but nothing response and next time I will shutdown by forcing and again trying to start my laptop and next I will seen the problem in my laptop screen display does not working or nothing showing at display but laptop power is on position and cooling system fan was running on slowly ?
Now at that moment i have needs to be your advising, which was component damaging /Replacing /Driver installing by removing this problem Kindly help me.
Waiting for your response as soonest.
Best Regards,
Md. Shariful Alam (Pallab)
Global Trade And Engineering.
Dhaka, Bangladesh.


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Oman Telecommunications Company (Omantel) substantially reduced monthly subscription charges and the SIM card rate as of January 1, 2000, according to Times of Oman daily. Furthermore, connection charges and feature set-up charges have been abolished for GSM subscribers. Price cuts have thus far included GSM subscription, which were cut by roughly 33 percent from RO6 to RO4 and a 53 percent SIM card rate cut from RO15 to RO7. (RO1=$2.6)
Other scheduled reductions include:
* Services, such as mailbox-to-mailbox message deposit and message notification (via pager, PSTN and GSM), are to soon be free.
* The price for monthly voice mail subscriptions for GSM users will be free and for others, reduced 50 percent from 500 baiza to 250 baiza.
* Monthly voice male subscription in the executive category will be substantially slashed.
* Monthly voice mails subscription for GSM and others will be cut from RO2 to 500 baiza.
Omantel has also reduced telephone rates for calls to Arab Gulf Cooperation Council countries, reduced data-service charges for GSM users from RO6 to RO3 and instituted discounts for Internet users.
Meanwhile, the country is offering new online Internet services, including billing, for Internet subscribers, according to the Oman Observer daily. The new on-line service -- developed by Tariq bin Hilal al Barwaini and supported by Omantel - allows Internet users to see how many hours they have spent on the Internet and check their billing status. This can all be done at http://www.tariq.net target
The site also offers others services such as the option to leave message on the server that will be sent to all incoming emails while a user is, for example, away on vacation and unable to personally respond. Another service is the ability to apply for an Internet connection.
© 2000 Al Bawaba (www.albawaba.com)


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2015-2016 Academic Year
UAB Information Technology strives to provide consistent, accessible, personalized and collaborative environments to enhance the student experience. The quicklinks below provide a 'get-started' list of the most common services students ask about. For even more information and resources available to the student community visit uab.edu/it/home/students.
BlazerID and Password
Most of UAB's secured online resources require you to log in using your BlazerID and password. Get a BlazerID or change your password at BlazerID Central at uab.edu/blazerid. Student passwords will expire every 180 days; you will receive notices when it is time to change it. To change or reset a forgotten password, visit BlazerID Central.
Using your BlazerID and password, log into BlazerNet, UAB's student portal - uab.edu/blazernet. From there you will be able to link to class registration, grades, financial aid and other academic resources.
Free software
Your Office 365 account gives you access to free Microsoft Office software, including Word, Excel and PowerPoint, along with 1 TB of storage in a OneDrive account. Other software is also available for free or at a discount.
Free software includes Office 365, Mathematica, Matlab, Endnote, DreamSpark, Antivirus software and Gartner research.
Discounted software includes Adobe Creative Cloud and Dell products.
Every student has access to a free 50 GB e-mail account through Office 365. Your UAB e-mail address
(firstname.lastname@example.com) is the place to look for all official correspondence from UAB. To check or update your e-mail preferences, visit BlazerID Central at uab.edu/blazerid. To register a UAB account, click "Get A UAB Email Account" on the left side of the web page.
Get help
For support with all things IT contact AskIT, UAB's IT help desk. Many self-help articles and FAQs are available on the AskIT website. To access the FAQs, open a ticket or chat with an agent, visit uab.edu/askit, or call AskIT at 205-996-5555. Walk-in help is also available at the Center for Teaching and Learning. Hours of operation vary and are posted on the AskIT website.
File Size & UAB Dropbox
Your Office 365 mailbox has 50 GB of space, and the size limit for attachments is 30MB. Large attachments should be sent via the UAB Drop Box at uab.edu/dropbox.
Wireless Networks
Stay connected on campus by accessing the UAB WiFi network using your BlazerID and password: uab.edu/it/wifi. For information about Apogee ResNet in the dorms, click here.
Mobile Users
Have an Apple or Android mobile device? Download the free UAB Mobile app which provides access to class schedules, maps, Campus Directory, news and athletics, and Canvas Mobile: uab.edu/app.
Online Classroom/Canvas
Online courses are available via Canvas (log in with your BlazerID and password at uab.edu/canvas). Training and support opportunities are available at the site.
Recommended System Specifications & Computer Setup
UAB IT provides recommendations on computer specifications for Apple Macintosh and Microsoft Windows PC laptop and desktop systems, and guidance on how to prepare your computer for use at UAB (before you arrive on campus). Visit uab.edu/it/students and select from the available resources for more detail.
Software & Computers
UAB IT offers an extensive library of software for free or at discounted pricing, including antivirus software, Microsoft Office products and more. Microsoft Office products are available to students for free through their Office 365 accounts, along with free storage on OneDrive. Through UAB's relationship with Dell, many computer products are available at a discount to students (and parents on behalf of students). Visit uab.edu/it/students and select from the available resources for more details.
Computer Labs
UAB maintains several student computing labs which are open to all current students with a valid BlazerID. For locations and hours, visit uab.edu/it/labs.
Campus Directory
The online directory contains contact information for students, faculty, staff and all UAB entities: uab.edu/directory.
Copyright/Download rules
The Higher Education Opportunity Act (HEOA) includes provisions to reduce the illegal uploading and downloading of copyrighted works through peer-to-peer (P2P) file sharing of movies, videos, music, etc. More information, including how UAB detects and responds to illegal downloading activity, can be found at uab.edu/it/heoa.
IT Policies
Individuals who use UAB technology resources are required to read the Acceptable Use Policy in its entirety. This and other IT policies, standards and best practices can be found on the UAB IT website: uab.edu/it/policies.
Students can register their cell phone for various notifications from around campus. From notifications about postings in the learning management system, to reminders about registration deadlines, to being able to reset your BlazerID password remotely (24x7)...it all starts with registering your cell phone and selecting which services you want to take advantage of. Visit uab.edu/enotify and sign up today.


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Paddling for several hours a day, so first of all you will have to expect physical strain. However, the sore muscles are worth it when you get to experience: fast flowing waters that make your adrenaline rush, untouched nature, wild animals such as: fishes, ducks, ... fresh air and a lot of cascades and rapids. All pretty culm and not dangerous.
The time to raft like a Robinson?!
The best time for rafting is between june 1. and September 15. In this period the nights are tolerable and the water is ideal for swimming. It is also the time when nature is most "alive" and there is plenty to see. Recommended duration of trip is 3 days but you can also choose up to 10 days river adventure.
|Map Legend
|Multy active week on river Kupa
Trg Kralja Tomislava 25, Brod na Kupi, Croatia


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Attached to the letter was an invoice of what was in the box.
Notice the circled item on the invoice: "Personalized Thank You Letter"
This just makes me feel SO appreciated.
(click to enlarge)
A Blog detailing the insanity of my medical practice and the stupidity of everyday life.
Im the one that takes care of the samples at my office, when i get levaquin from triple i, it looks just like this. one day, my shipment came in and it said "levaquin-dr information sheet," it was a piece of paper that literaly said "levaquin 360" that was it....
Dear Grumpy,
I'm so totally psyched that you chose to hit us up hardcore for some Axert up in this bitch. I sent out a group text to like Joe in shipping, Bill in accounting, as well as Shelly the receptionist to let 'em know too and, dammit, whaddya know, they were all as psyched as I that you hit us up! We all read your blog, and it's hot like wasabi to say the least. It's totally wicked, dood, seriously.
We at Triple i wish to extend you our sincerest double-dose of gratitude for promoting our product via free samples. I hope you find out what we already know: AXERT RULZ and IMITREX IS FO CHUMPS!!!!!!!!
You bess be prescribin'!
--Triple i
It does make one feel oh so extra special doesn't it?
Glad they have a good system for their packers to not forget the letter. Think of how you would have felt not getting one. LOL
That is AWESOME, Nurse K.
Was there any thought to not signing the confirmation sheet so they would have to send you another personalized thank you letter and have you sign for it again?
No, because I hate it when I get the 2nd letter. It's often registered, and some companies now even require a notary since a C drug is involved.
So I just sign the first one and go on my happy way.
Little Calf Creamery. Delicious homemade ice cream and food in Thousand Oaks!


text:
About Us
Our Company
Insurance is our only business. Affinity groups are our only clients, and we've managed insurance programs for some of them for more than four decades. We are passionate about service, and design all of our products and procedures in the best interests of our customers.
We design, sell, and manage specialty insurance programs, endorsed by organizations exclusively for members and their families. As experienced administrators, we've combined decades of insurance knowledge with cutting-edge product and marketing innovations. Insurance is still our only business, but we've taken it in many new directions.
Since 2004, we've more than doubled in size and quadrupled our benefits and services. We're extremely proud that our growth has been 100% organic, not through outside funding, merger, or acquisition, so we've been able to maintain the continuity and personal attention our clients and customers have come to expect.


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Kimmirut, Nunavut Territory, Canada
(1973- )
Simeonie's father was artist Shorty Killiktee. Simeonie has exhibited in Canada, the United States and Germany.
Spirit Wrestler Gallery
101-1669 West 3rd Ave.
Vancouver, BC
Canada V6J 1K1
Toll Free: 1-888-669-8813
Phone: 604-669-8813
one block West of the Granville Island gates
Between Pine St. and Fir St.
Tuesday to Saturday, open 10-5
Sunday and Holidays, open 12-5
© 2019 Spirit Wrestler Gallery. All Rights Reserved.


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We invite you to JOIN our 100% free forum today so that you can post and interact with our members. Registration is quick and easy, so REGISTER today!
We look forward to seeing you as a member soon!
Come talk about VHF and UHF equipment and operation
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Today in London, Christie's auction house sold a rare first edition of Charles Darwin's On the Origin of Species as part of the celebration of the 150th anniversary of its first publication. The book, one of only 1,250 copies first printed in 1859, was found on a shelf in a family's guest bathroom in Oxford. Christie's said they thought the book would likely sell for around $100,000, but an anonymous telephone bidder won the green bound, gilt-decorated tome for about $171,000. Darwin's Origin of Species outlined his theory of natural selection and is the foundation for the modern understanding of evolution.
Of course, many fearful zealots thoroughly believe, and joyfully so, that Charles Darwin is now barbequing away, racked in hideous, throbbing pain, burning forever in the pit of hell for the rest of eternity because of his blasphemous thought provoking ideas, such as his belief that man evolved from lower forms of primates and random mutation.
Many of the least thinking biblical pundits and dim ostriches would like to blame Darwin and all his followers for the reason why this good christian nation is sliding into all things wicked. Any preacher worth his fear mongering salt will certainly lecture on the evil Stalinism these monkey loving evolutionists are shoving down the throats of the faithful joes and janes, taking society to hell in a hand basket with their flu shots, homosexuality, flag burning, pro-choice, science education, gun control, cunnilingus, hip hop, global warming, masturbating, MSNBC, health care reform.
Fear not creationists, god created Kansas just for you. Do I hear an amen!?


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Canis_lupus wrote:I think TotQ should be out of the HO loop just because its doesnt discuss Politics like the other 2. but its not my call.
rbos wrote:Putting politics into TotQ means that the forum is fair game for political responses. Unless you plan to separate your political comic strips from your real comics, I don't see anything wrong with your readers assuming the same.
Tom Mazanec wrote:The phrase is actually "eat your cake and have it too." (my little English language quirk...I know the phrase is being corrupted in the vernacular).
Tom Mazanec wrote:Skull:
Buy the CD this summer.
How many times have you found a favorite artist, writer, musician, comedian, actor, etc. only to discover, in an ugly October Surprise, that the artist you'd been supporting with your trade for years held beliefs or practices you found unimaginably repugnant? It's an incredibly nasty feeling to realize your consumer dollars had been financing their lifestyle and agenda, isn't it? Made you feel mad and betrayed, didn't it? Felt a little bit deceived, didn't you?
To Skull, I have but one thing to say:
"Door's to your left."
Canis_lupus wrote:Its been proven that if you use constructive criticism from your audience you will be hard pressed to become profitable. Using advice given to you is not showing weakness.
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Google won't join Wikipedia in going black at midnight on Wednesday but the Mountain View-based search company will join a digital protest against SOPA.
The controversial Congressional measure, which would give the US Justice Department the ability to track and go after Internet pirates, has rallied some of Silicon Valley's largest tech companies together to oppose the measure.
Google is joined by Twitter, Yahoo, Facebook, Craigslist, LinkedIn, eBay, AOL, Zynga, Wikipedia and more.
Wikipedia had tried to organize those tech companies in opposition to go dark for 24 hours Wednesday but instead Google will add a link to its homepage where users will be able to learn more about the measure.


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First there was the Buzzcocks shilling for Toyota. Uhm, no. Camping and Sport Utes have not ever been, nor will they ever be, punk rock. A beat up '91 Corolla plastered in xerox stickers and missing its hubcaps is punk rock, but camping? No. Next comes Iggy Pop croaking away for Cadillac with Teddy Bears. Closer! Detroit and Iggy do indeed have a solid connection after all. While these two examples of corporate adoption of punk rock heroes are vaguely disturbing, there is one musical association with automobiles that we would like to see go away now and forever. Please make Sheryl Crow stop whining on about winding roads for Subaru. With that, we serve up this '70s-era Subaru Leone commercial. Groovy musical artist unknown.
The Taming of the 'Ru: 1973 Subie Ad; It's All Toyota's Fault: Everybody's Ancient Nowadays [Internal]


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DiS brings you the first in a series of exclusive video blogs from the Northern Alliance Tour featuring Rolo Tomassi, Grammatics and Pulled Apart By Horses, a little later than originally scheduled.
Your narrator, James Brown from Pulled Apart By Horses, had a few problems with his leg - he was hospitalised shortly before the tour, and his band missed the first few dates (and filming opportunities) as a result. Thankfully, he's feeling better now.
We join the bands in Liverpool for the first instalment - those of a nervous disposition, or with an aversion to seeing topless, sweaty indie boys, should think carefully before hitting 'Play'.
The dates for this tour are as follows:
14 Leicester The Musician
15 Sheffield The Plug
16 Wrexham Central Station
17 Nottingham Bodega Social
18 Manchester Satans Hollow
19 Leeds The Faversham (no Grammatics)
20 Middlesborough Uncle Alberts
21 Liverpool Barfly
22 Glasgow Stereo
28 Bristol Croft
29 Cardiff Barfly
30 Exeter Cavern (no Pulled Apart By Horses)
1 Bournemouth Gander
3 Southampton Joiners
4 Oxford Academy 3


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An easy shelving project is the solution for the boxes of books piling up in the house, writes Justin Newcombe.
I love owning books. For many years I have been able to house only a small portion of my library, the bulk of the rest of it has been allocated to cardboard boxes. Sadly, in recent times I've had to cut my book habit down just because of a lack of storage. I suppose a Kindle may soon be on the cards, however, the book as an object, its weight, its artwork, the nuances of text and format are all things which are not easily substituted, which is why I'll always buy them.
A bookcase is something you can easily build with little fuss. I've gone for a plain pine finish on this because it's a bit understated. The interest is in the books, not the case. But if you want to paint yours, use MDF instead of pine. Bunnings have both MDF and clear pine already cut into convenient lengths, making transport and handling easy. I used a biscuit joiner, which cuts little slots in the end of the timber so you can insert a small, elliptical-shaped dowel. This makes the joins strong and then there's no need for screws.
Step 1
Work out the height and width of your bookcase then divide the height measurement with the number of shelves you want. This will give you the distance between each shelf.
Step 2
Cut the top, bottom and sides of the bookcase to length. Place the side panels on to a flat surface, side by side and perfectly flush at each end.
Step 3
Mark and drill the shelving-pin holes. Use a tape measure first to measure out where each shelf will go. Mark it, then run a large set square across the measurement to keep the lines exactly square. Place a mark 30mm inside each edge on both pieces of timber. Repeat for each shelf.
Step 4
Carefully drill out the shelving-pin holes. Use the drill bit size recommended on the packet of shelving pins. Be careful not to drill right through the timber. It is worth investing in a small depth gauge, available at Bunnings, which attaches to the drill bit to restrict the depth of the hole.
Step 5
To inset the biscuit joiners, mark out the ends of the wall sections 100mm inside the side then draw a vertical line approximately 100mm long. Adjust the biscuit cutter so the blade is positioned at exactly half the width of the timber at a cutting depth half the width of the joining biscuit you're using. Do a test cut and make any final adjustments. Line up the biscuit cutter with the vertical line and carefully make the cut into each end of the side timbers.
Step 6
For the top and bottom piece, mark out a line on each end, 100mm inside each side. To check your measurements, do a test using an off-cut by inserting a biscuit into it and assembling it with one of the wall sections.
Once you're satisfied, carefully make the cut with the biscuit cutter. The cut for the top and bottom will be on the face of the timber rather than in the end.
Step 7
Glue in the biscuit and assemble the shelves. I used right angle clamps to get the whole thing square.
Step 8
Glue and screw the backing panel. I used two sections of 4mm marine ply, but 6mm MDF is fine.
Step 9
Measure and cut the shelves. Sand the bookcase and shelving. I used 250-grit sandpaper and a sanding sponge, which is good for softening hard edges.
Step 10
Insert the shelving pins, place into position and insert each shelf.