Suitable bioconjugation strategies and stabilisation of biomolecules on electrodes is essential for the development of novel and commercially viable biosensors. In the present review, the functional groups that comprise the selectable targets for practical bioconjugation methods are discussed. We focus on describing the most common immobilisation techniques used in biosensor construction, which are classified into irreversible and reversible methods. Concerning the stability of proteins, the two main types of stability may be defined as (i) storage or shelf stability, and (ii) operational stability. Both types of stability are explained, as well as the introduction of an electrophoretic technique for predicting protein–polymer interactions. In addition, solution and dry stabilisation as well as stabilisation using the covalent immobilisation of proteins are discussed including possible factors that influence stability. Finally, the integration of nanomaterials, such as magnetic particles, with protein immobilisation is discussed in relation to protein stability studies.

The mechanisms underlying the accelerated decline of the intracellular Ca2+ transient that occurs in cardiac muscle when stimulation rate is increased have been investigated in ventricular myocytes from rat hearts. Increasing stimulation rate from 0.1 to 0.5 and 1 Hz decreased the time taken for the Ca2+ transient to decline from its peak to 50% of its peak value in cells generating action potentials, when the duration of depolarization was held constant by voltage clamp, and when Na/Ca exchange was inhibited. The sarcoplasmic reticulum Ca2+ adenosinetriphosphatase inhibitor thapsigargin inhibited rate-dependent abbreviation of the Ca2+ transient. However, neither a chemical inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (KN62) nor a peptide inhibitor of this enzyme (calmodulin-binding domain peptide) had a significant effect on rate-dependent abbreviation of the Ca2+ transient. Analysis of the phosphorylation of the regulatory sites Ser16 and Thr17 of phospholamban showed no significant change in phosphorylation with changes of stimulation rate. These data suggest that rate-dependent shortening of the Ca2+ transient is due predominantly to enhanced Ca2+ uptake by the sarcoplasmic reticulum without changes in phospholamban phosphorylation.

HighlightsA modified glassy carbon electrochemical sensor for microRNAs was developed.The electrode allowed detection of femtomolar concentrations of miR-21.The method was applied to detection of urinary miR-21.

Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1±20), interacts with the Ca 2+ ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1±20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1±20). PLB(1±20) is shown to interact with a peptide corresponding to residues 378±405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1±20 of PLB. They also suggest that phospholamban residues 1±20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378±405).Keywords: phospholamban; calcium-activated ATPase; sarcoplasmic reticulum; phosphorylation; NMR.The Ca 2+ ATPase of the sarcoplasmic reticulum (SERCA) is a calcium pump which functions in association with the Ca 2+ release channels, and is responsible for maintaining the calcium transients upon which the regulation of contractile activity in all types of muscle depends. The SERCA enzyme family consists of a number of isoforms encoded by three genes that are expressed in a tissue-specific manner. In cardiac muscle the isoform characteristic of that tissue, SERCA2a, is associated with phospholamban (PLB), a 52-residue polypeptide whose reversible phosphorylation enables the calcium pump to be modulated [1,2]. Interestingly, the same two proteins are expressed in slow-twitch skeletal muscle [3,4], whereas different isoforms, SERCA1a and 1b [5] but no PLB [6] are present in fast-twitch skeletal muscle. Nevertheless, SERCA isolated from fast skeletal muscle can be regulated by PLB in vitro [7±9], although it has been suggested that it is regulated in situ by a related protein, sarcolipin [10].The presence of PLB enables the calcium pumping rate of the sarcoplasmic reticulum to be increased in cardiac, and to a lesser extent in slow-twitch skeletal muscles, in response to b-adrenergic stimulation. Although the detailed mechanism of this process is not understood, a current view is that in the unphosphorylated form PLB interacts with SERCA and inhibits pumping activity, possibly by reducing its affinity for calcium ions (compare [11] with [12,13]), and reducing the maximum turnover rate at saturating calcium ion concentration, which is reflected in the Ca/MgATPase activity rate (reviewed in [14,15]). Phosphorylation of Ser16 of PLB by cAMP or cGMP dependent protein kinases and/or phosphorylation of Thr17 by the multifunctional calmodulin dependent protein kinase [16,17] results in increased activity of the pump [18,19].The detailed mechanism by which PLB m...

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