Incorporation Of Linkers Research Articles

Evaluation of Linkers' Influence on Peptide-Based Piezoelectric Biosensors' Sensitivity to Aldehydes in the Gas Phase.

Recent findings qualified aldehydes as potential biomarkers for disease diagnosis. One of the possibilities is to use electrochemical biosensors in point-of-care (PoC), but these need further development to overcome some limitations. Currently, the primary goal is to enhance their metrological parameters in terms of sensitivity and selectivity. Previous findings indicate that peptide OBPP4 (KLLFDSLTDLKKKMSEC-NH2) is a promising candidate for further development of aldehyde-sensitive biosensors. To increase the affinity of a receptor layer to long-chain aldehydes, a structure stabilization of the peptide active site via the incorporation of different linkers was studied. Indeed, the incorporation of linkers improved sensitivity to and binding of aldehydes in comparison to that of the original peptide-based biosensor. The tendency to adopt disordered structures was diminished owing to the implementation of suitable linkers. Therefore, to improve the metrological characteristics of peptide-based piezoelectric biosensors, linkers were added at the C-terminus of OBPP4 peptide (KLLFDSLTDLKKKMSE-linker-C-NH2). Those linkers consist of proteinogenic amino acids from group one: glycine, L-proline, L-serine, and non proteinogenic amino acids from group two: β-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid. Linkers were evaluated with in silico studies, followed by experimental verification. All studied linkers enhanced the detection of aldehydes in the gas phase. The highest difference in frequency (60 Hz, nonanal) was observed between original peptide-based biosensors and ones based on peptides modified with the GSGSGS linker. It allowed evaluation of the limit of detection for nonanal at the level of 2 ppm, which is nine times lower than that of the original peptide. The highest sensitivity values were also obtained for the GSGSGS linker: 0.3312, 0.4281, and 0.4676 Hz/ppm for pentanal, octanal, and nonanal, respectively. An order of magnitude increase in sensitivity was observed for the six linkers used. Generally, the linker's rigidity and the number of amino acid residues are much more essential for biosensors' metrological characteristics than the amino acid sequence itself. It was found that the longer the linkers, the better the effect on docking efficiency.

ProtaTETHER: A method for the incorporation of linkers in biomacromolecules.

The impacts of linkers on dynamics, expression, and activity of biomacromolecules are often overlooked. This may be due, in part, to the lack of facile methods for incorporation and analysis of linkers that vary iteratively in both length and sequence composition. The protaTETHER method addresses this gap by enabling the incorporation of focused linker libraries at potentially any region in a protein sequence. In this chapter, we describe the generation and incorporation of linkers in a PKAc-GFP fusion protein and provide methods for the application and evaluation of the protaTETHER process.

Hierarchical Synthesis, Structure, and Photophysical Properties of Gallium- and Ruthenium-Porphyrins with Axially Bonded Azo Ligands.

The hierarchical synthesis of three porphyrin and four bisporphyrin derivatives is presented. This strategy relies on the incorporation of linkers based on azo moieties appended with pyridyl and/or acetylenic groups that facilitate axial coordination to Ga- and Ru-metalloporphyrins. These porphyrinic systems allow for a quantitative analysis of the effects of diamagnetic anisotropy (DA) by using 1 H NMR spectroscopic and X-ray crystallographic analyses. A simple power-law relationship between the proton chemical shift and the distance from the porphyrin core is experimentally outlined, which confirms previous theoretical predictions and shows that the limit of DA is about 2 nm. Photophysical properties of the azo-linked porphyrins are analyzed by UV/Vis spectroscopy, showing that significant cis-trans isomerization is not observed for azo ligands bound only to Ga-porphyrins. Incorporation of Ru-porphyrins to an azo ligand facilitates photoswitching behavior, but the process faces competition from decarbonylation of the Ru-porphyrin, and appreciable switching is only documented for GaL1Ru.

Azulene-based conjugated polymers: unique seven-membered ring connectivity leading to stimuli-responsiveness

A novel family of stimuli-responsible polyazulenes connected through the seven-membered ring, in contrast to traditional five-membered connectivity, have been prepared by Yamamoto-, Sonogashira-, and Stille-coupling of 4,7-dibromoazulenes. Polymer absorption can be tuned by structural modification of the conjugated backbones with UV-vis and EPR studies indicating that the seven-membered ring connection of the azulene unit is effective for the generation and stabilization of azulenium cations. The degree of conjugation and the reversibility of optical properties upon protonation and deprotonation is sensitive to the steric environment of the seven-membered ring and can be tuned by both substitution and incorporation of linkers between the azulene units.

INFLUENCE OF DIFFERENT LENGTH LINKER CONTAINING DHEA-7-CMO-ENZYME CONJUGATES ON SENSITIVITY AND SPECIFICITY OF DHEA-17-CMO-ANTIBODY

Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime–bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates.

Incorporation of Different Bridge Length Linkers in Enzyme and Its Use in the Preparation of Enzyme Conjugates for Immunoassay

An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6‐amino caproic acid (6ACA) followed by ADH. The different bridge length linkers‐incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol‐21‐hemisuccinate–HRP (cortisol‐21‐HS–HRP), cortisol‐21‐HS–ADH–HRP, cortisol‐21‐HS–ADH–GABA–HRP, and cortisol‐21‐HS–ADH–6ACA–HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay.

An efficient method for generation and subcloning of tandemly repeated DNA sequences with defined length, orientation and spacing.

Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.