Solid phases/substrates

Classic solid phase substrates used in biotesting, such as microtiter plates, membrane filters or microscope slides, have been the first supports used for NA immobilization in array fabrication [27]. Desired attributes of any DNA array substrate include (i) chemical homogeneity (ii) thermal and chemical stability (iii) ability to control surface chemical properties such as polarity or hydrophobicity (iv) ability to be activated with a wide range of chemical functionalities (v) reproducibihty of the surface modification processes involved (vi) inert with respect to enzymatic activity especially ones involved in DNA manipulation and (vii) ultra-low intrinsic fluorescence. 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

Based on an extensive theoretical treatment of equilibrium positions,P l an interesting approach of solid-to-solid conversion catalyzed by proteases has been developed. According to this, a favorable equilibrium shift toward the peptide product is given when the starting reactants are largely undissolved in the reaction medium and the product precipitates. The use of solid-phase substrate pools combines the equimolar or nearly equimolar supply of reactants with high obtainable yields, easy workup procedures and, in principle, compatibility with conventional chemical peptide synthesis standard procedures. Both the advantage of solid-phase substrate pools mainly in equilibrium-controlled synthesisl " and the extension of this approach to protease-catalyzed acyl-transfer reactionst have been successfully demonstrated. 

Various biochemical methods are available for measuring heparanase and other glycosidase activities. The former can be assayed by a method that makes use of a solid-phase substrate for heparanase, such as chemically-modified heparan sulphate coupled covalently at its reducing terminal saccharide to agarose gel beads. The reader is referred to Nakagima et al. (1986). 

Nakagima, M., Irimura, T. and Nicolson, G. L. (1986). A solid-phase substrate of heparanase its application to assay of human melanoma for heparan sulfate degradative activity. Anal. Biochem. 157, 162-171. 

Figure 12.5-15. General principle of application of equilibrium shift towards the product by solid-phase substrate pools (botton) compared with synthesis starting from solution11721.

Lateral density fluctuations are mostly confined to the adsorbed water layer. The lateral density distributions are conveniently characterized by scatter plots of oxygen coordinates in the surface plane. Fig. 6 shows such scatter plots of water molecules in the first (left) and second layer (right) near the Hg(l 11) surface. Here, a dot is plotted at the oxygen atom position at intervals of 0.1 ps. In the first layer, the oxygen distribution clearly shows the structure of the substrate lattice. In the second layer, the distribution is almost isotropic. In the first layer, the oxygen motion is predominantly oscillatory rather than diffusive. The self-diffusion coefficient in the adsorbate layer is strongly reduced compared to the second or third layer [127]. The data in Fig. 6 are qualitatively similar to those obtained in the group of Berkowitz and coworkers [62,128-130]. These authors compared the structure near Pt(lOO) and Pt(lll) in detail and also noted that the motion of water in the first layer is oscillatory about equilibrium positions and thus characteristic of a solid phase, while the motion in the second layer has more... 

The adaptation of the Bischler-Napieralski reaction to solid-phase synthesis has been described independently by two different groups. Meutermans reported the transformation of Merrifield resin-bound phenylalanine derivatives 32 to dihydroisoquinolines 33 in the presence of POCI3. The products 34 were liberated from the support using mixtures of HF/p-cresol. In contrast, Kunzer conducted solid-phase Bischler-Napieralski reactions on a 2-hydroxyethyl polystyrene support using the aromatic ring of the substrate 35 as a point of attachment to the resin. The cyclized products 36 were cleaved from the support by reaction with i-butylamine or n-pentylamine to afford 37. 

Solid-phase synthesis (Section 26.8) A technique of synthesis whereby the starting material is covalently bound to a solid polymer bead and reactions are carried out on the bound substrate. After the desired transformations have been effected, the product is cleaved from the polymer. 

A short and efficient synthetic approach to hydroxy-substituted ( )-stil-benoids, as exemplified by the natural compound resveratrol (371b) via solid-phase CM, was reported by a Korean group (Scheme 71) [154]. When two different stilbenes were allowed to couple by catalyst C, all three kinds of possible stilbenes were obtained as an inseparable mixture. Anchoring 4-vinylphenol to Merrifield resin, followed by exposing the supported styrenyl ether 368 and diacetoxy styrene 369 (10 equiv) to the catalyst, inhibited self-metathesis of the supported substrate. Sequential separation of the homodimer formed from 369 by washing and subsequent cleavage of the resin 370 with acid provided (E)-stilbene 371a with complete stereocontrol in 61% yield. 

The biberty (Fig. 10), a monomode microwave reactor for automated SPPS, was recently introduced by the CEM Corporation [153]. Although this instrument was originally developed for SPPS, it also allows for a broader scale of solid-phase applications. The solid-phase vial is equipped with a polypropylene frit and cap at one end (the entire assembly fitting into the standard 10 mb CEM reaction vessel) to allow the processing of 0.1 to 1.0 mmol quantities of resin attached substrates. An integrated fiber optic probe provides... 

The possibility of multiple reactions and the number and composition of possible solid phases, with the inclusion of the substrate as a possible reactant. 

These reactors contain suspended solid particles. A discontinuous gas phase is sparged into the reactor. Coal liquefaction is an example where the solid is consumed by the reaction. The three phases are hydrogen, a hydrocarbon-solvent/ product mixture, and solid coal. Microbial cells immobilized on a particulate substrate are an example of a three-phase system where the slurried phase is catalytic. The liquid phase is water that contains the organic substrate. The gas phase supplies oxygen and removes carbon dioxide. The solid phase consists of microbial cells grown on the surface of a nonconsumable solid such as activated carbon. 

Figure 2 Immobilized antigen ELISA format. Antigen is immobilized to a solid phase by passive adsorption. Following removal of unbound antigen, analyte (free H) and antigen (H-protein) compete for a fixed number of primary antibody (Y) binding sites. Unbound materials are removed (dotted line). Secondary antibody-enzyme conjugate (Y-E) is added to bind to primary antibody followed by another wash step. Substrate (A) for the enzyme is added to detect the bound enzyme. The amount of colored product ( ) detected is inversely proportional to the amount of analyte present...

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

Figure 1 Schematic of an enzyme immunoassay. (1, 2) The test solution and enzyme conjugate are added to a tube or well pre-coated with anti-anal) e antibodies. (3) After the inhibition step, the solid phase is washed, and only antibody-bound material is retained. (4A-C) Colorless substrate is added and is converted to a visible color in inverse proportion to the amount of analyte in the sample...

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