Redox labeling

Determination of the thermodynamic and kinetic parameters of interest requires monitoring of the surface concentration of the binding molecule. With large biomolecules, the surface concentrations are small, and simple redox labeling will not allow sufficient sensitivity. Labeling of the target biomolecule with a redox enzyme obviates this difficulty, thanks to the catalytic properties of the enzyme. 

A more recent application of redox labeled ODNs is redox-active aptamers that exploit molecular recognition between the aptamer and a target analyte. Briefly, aptamers are functional nucleic acids that selectively bind to a variety of targets. Due to a well-defined three-dimensional structure, aptamers can achieve selectivity comparable to that of antibodies but are readily accessible taking advantage of well-known nucleic acid chemistry, polymeric chain reaction and contemporary separation methods, followed by aptamer selection from random pools of nucleic acids (DNA or RNA) by in vitro selection process called systematic evolution of ligands by... 

The formation of aptamer-substrate complexes was also followed by the use of redox-active intercalators73 (Fig. 12.18d). A nucleic acid hairpin structure that contained in its single-stranded loop the antithrombin base sequence was assembled on a Au electrode, and methylene blue was intercalated as a redox label in the double-stranded stem of the hairpin structure. The hairpin was, then, opened in the presence of thrombin, by generating the respective G-quadruplex-thrombin complex, and as a result, the redox label was removed from the nucleic structure, showing a decrease in the voltammetric response with the increase in the concentration of thrombin. This method enabled the analysis of thrombin with a detection limit that corresponded to... 

Another method for the analysis of aptamer-protein complexes involved the use of a positively charged ferrocene-tethered polythiophene, (19), as redox label reporting unit (Fig. 12.19). The antithrombin aptamer was immobilized on an electrode surface, and the electrostatic binding of the redox polymer (19) to the aptamer monolayer resulted in a supramolecular complex that revealed electrical contact between the polymer and the electrode.74 The formation of the aptamer-thrombin complex removed the polymer from the surface and blocked the electrical contact between the polymer label and the electrode. As a result, higher concentrations of thrombin increased the surface coverage of the aptamer-thrombin complex on the electrode, and this decreased the amperometric responses of the sensing device. 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

Single-nucleotide polymorphisms (SNPs) have been analysed on a capillary gel electrophoresis (CGE) microchip with EC detection [150]. The genetic section that contained the SNP was amplified by PCR and purified. Then, it was used in a single-base extension (SBE) reaction with a redox-labeled chain terminator, ferrocene-acycloATP. Products of the SBE, ferrocene-labeled SNP and free ferrocene-acycloATP, were separated employing CGE on microchip and detected using sinusoidal voltammetric detection at a pyrolysed photoresist film (PPF) electrode. 

Scheme 13 Electronic and optical transduction of the formation of antigen-antibody affinity complexes on transducers (A) ampero-metric transduction at an electrode (R+/Ris a redox label in the electrolyte solution), (B)...

UV photolysis of CpMn(CO)3 in toluene leads to loss of CO and formation of CpMn(CO)2( ] -toluene). Kinetic studies suggest that the binding energy of the toluene is ca. 60kJmor. The binding of H2 to CpMn(CO)2 has been studied in supercritical CO2 solvent. It has been proposed that pyrylium and pyridinium salts such as (35) can be used to label proteins and thereby aid in the detection and characterization of receptor sites. Cymantrene bound to lysine residues of bovine serum albumin (BSA) has been used as a redox label. Electrochemical reduction of the label established an impressive BSA detection limit of 2 x 10 M. 

Ruthenium has been incorporated into ODNs, via an oxime conjugate,or through an amide linker. In the latter case, the ruthenium conjugate was used to photo-crosslink with a G residue on a complementary strand. Ruthenium and osmium have been incorporated into ODNs via a C5-phenanthroline dU amidite to study photoinduced energy transfer, and metallocarboranes have been used as redox labels by attachment to 04 of dT. Ferrocene has... 

Le Gal La Salle, A. Limoges, B. Rapicault, S. Degrand, C. Brossier, P. New immunoassay techniques using Nafion-modified electrodes and cationic redox labels or enzyme labels. Anal. Chim. Acta 1995, 311, 301-308. 

Di Gloria et al. [54] proposed the use of electrode-enzyme coupling for the amplified readout of a redox label, using a lidocaine-ferrocene conjugate in combination with GOx. The use of ferrocene as a mediator for glucose determination within GOx enzyme electrodes provided the... 

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