Covalent attachment of probes

A covalent bond is formed by the sharing of electrons between two atoms. The dissociation energy for a typical covalent bond is 100 kcal/mol and by far the strongest in biochemistry. One can distinguish between a covalent attachment of activated probes and a covalent attachment of probes on activated surfaces. 

Fluorescence resonance energy transfer (FRET) is a powerful method for the determination of macromolecular conformation and folding in solution [188, 190-194]. The method relies on the incorporation into the macromolecule of a donor dye molecule and an acceptor dye molecule that undergo singlet-singlet Forster energy transfer, which is distance dependent over the 10-100 A range [190-194]. Early work in the field focused on probes noncovalently associated with the macromolecule, but covalent attachment of probes, while harder to do,... 

The interaction of ACh with the Torpedo nAChR. The data shown compare the equilibrium binding parameters obtained from fluorescence studies using covalently attached fluorescent probes and those obtained from radiolabelled [ H]ACh binding studies or functional measurements of cation flux. These data support a model in which the Torpedo nAChR carries sites of different affinities for ACh. We have previously suggested that occupancy of the lower affinity sites leads to channel activation whereas the higher affinity sites may play a role in desensitization processes... 

Dendrimers are branched chemical structures which can possess a range of terminal functionalities. Covalent attachment of dendrimers to a support builds a 3D structure along its surface which can subsequently be grafted with ligands which, as a consequence of the larger surface area, can yield higher probe densities. 

Figure 3.5 Comparison of adsorptive and covalent attachment of capture probes. (From Belosludtsev, Y. et al.. Anal. Biochem., 292, 250-256, 2001. With permission.)...

Belosludtsev and coworkers (2001) propose that the unmodified probes on the weakly cationic surface, although prevented from diffusing off the surface because of electrostatic interaction, nevertheless are available for hybrid nucleation. They suggest that such probe behavior could be viewed similarly to behavior observed in a liquid crystal matrix. This would be in sharp contrast to models describing the covalent attachment of short oligonucleotides as "oligo lawns" or monolayers of coiled probes (see Figure 3.6). 

The polymerization of substituted lactones is an attractive strategy for extending the range of aliphatic polyesters and for tailoring important properties such as biodegradation rate, bioadherence, crystallinity, hydrophilicity, and mechanical properties [100]. Moreover, the substituent can bear a functional group, which can be very useful for the covalent attachment of drugs, probes, or control units. 

Specifically, two different methods for the fabrication of these sensitive monolayers have been used. These methods differ in the technique used for the covalent attachment of the fluorescent probes (Fig. 2, step ii) while the first and the third fabrication steps (silanation (Fig. 2, step i) and binding molecule attachment (Fig. 2, step iii)) are identical for both methods. The first method is a solution-based procedure and the second method is a microcontact printing (/xCP) based procedure. In the solution-based procedure (Sect. 2.2.1), all... 

Krull and Wang [93] have reported preliminary work on the immobilization of oligonucleotide probes on the surface of an optical fibre where the probe molecules were functionalized by covalent attachment of intercalating dye. Probe sequences that were 20 nucleotides in length were covalently... 

A number of important conclusions were drawn from this study, as follows. Electrochemical reversibility in electroactive self-assembled monolayers depends upon concentration and polarity of a covalently attached redox probe. Reversible surface electrochemistry is observed for the well-diluted ferrocenyl ester. However, reversibility decreases with steric congestion of redox probe because higher redox probe concentrations lead to disorder due to cross-sectional mismatch of the redox probe and the alkyl chain. Reversibility also decreases with a nonpolar redox probe the alkylferrocene (System 4) yields broad peaks with long tails positive of E°, consistent with kinetic dispersion of the redox probes and their differential solvation in the SAM. 

Fig. 4 General approach of the bioreactive MALDI mass spectrometer probe tips. Goid piated probe tips are activated through the covalent attachment of enzymes (the general terminology of Au/enzyme is used to indicate the nature of the activated surfaces). The probe tips are then used for protein characterization by direct appiication of the analyte and time given for digestion. The digestions are stopped with the addition of a MALDI matrix, the reaction product-matrix mixture aiiowed to dry, and the probe tips are inserted into the mass spectrometer for MALDI-TOF analysis.

Scheme 4. Idealized sketches of gold surfaces for DNA hybridization sensors prepared by (a) chemisorption of thiolated ssDNA capture probes followed by chemisorption of a dilutor alkylthiol [168] and by (b) chemisorption of a mixed alkylthiol SAM followed by covalent attachment of an amino-terminated ssDNA capture probe (surface-bound carboxylic groups are activated by reaction with 0-(N-auccimrmdyl)-N,N,N, N -tetramethyluronium tetrafluoroborate in acetonitrile in the presence of N, A-diisopropylethylamine, [172]).

The present paper investigates various routes for covalent attachment of the tacn macrocycle to a pre-formed support matrix. Two different spacers are used to link the surface and the tacn propyl (P), and glycidoxypropyl (GP). The affinity of the modified surface for metals is probed with the test ions Cu and Mn2+, and styrene is the test substrate for the selective hydrocarbon oxidation. A preliminary note on this work has appeared [15]. 

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