Protein and Peptide Letters - Volume 15, Issue 8, 2008

Analyte detection by immobilized, biologically active macromolecules (biomolecules) has emerged as a highly effective technology serving the needs of both diagnostic industry and fundamental proteomic research [1]. The vast majority of immobilized biomolecules are proteins, very often enzymes or antibodies. Recently, the immobilization of DNA and RNA molecules has added a new array of applications to the biosensing technology [2]. Although important innovations have been made in the past ten years in order to build reliable biosensors, important challenges have yet to be met. Biomolecules immobilization needs to be simple, inexpensive, highly repeatable (especially in a manufacturing environment) and without interference with the biological processes being measured. Biomolecules need to sustain the harsh chemical treatments used during the immobilization process. The immobilized biomolecules also need to be stable (6 to 12 months at 4°C). This is a particularly delicate task especially when the requirement is to maintain the activity/the structural integrity of proteins in an artificial, semi-solid, environment that very often lacks adequate ionic strength, buffering power and anti-oxidant capabilities. Another key challenge encountered during the development of reliable biosensors is the efficiency of communication between the immobilized biomolecule (that serves as a receptor) and the inorganic material in its vicinity that plays the role of signal transducer. Tailored bio-interfaces and oriented immobilization are some of the key solutions to this challenge [3]. The selection of articles included in this special issue is aimed at presenting the recent advances in biomolecules immobilization, signal acquisition and signal processing. For this purpose, an illustrative variety of protein-based and nucleic acid-based sensors is described and discussed. A particular stress is put on the performance of the sensing device in terms of selectivity and sensitivity. The present special issue is composed of three reviews and five original research articles. The first review describes the main biomolecules immobilization strategies with an emphasis on oriented immobilization techniques (Prieto-Simon B. et al.). A second review (by Zhang D. et al.) details the design of protein-based voltammetric biosensors fabricated with nanomaterials. The third review (by Galban J. et al.) is dedicated to the detection of organic compounds using reagentless optical biosensors. The research articles included in this issue describe the development of new sensitive sensors for the detection of propionate (Sode, K. et al.), α-1-fetoprotein (Sun A-L. et al.), naphthalene acetic acid (Nikolelis D.P. et al.) and organophosphates and carbamates (Pohanka M. et al.) An original article by Hianik T. and co-workers reports on the development of biosensors based on aptabodies: artificial receptors formed by capping DNA aptamers with specific protein binding sites. REFERENCES [1] Choi, J.W., Oh, B.K., Kim, Y.K. and Min, J. (2008) J. Microbiol Biotechnol., 17, 5-14. [2] Lucarelli, F., Tombelli, S., Minunni, M., Marrazza, G. and Mascini, M. (2008) Anal. Chim. Acta., 609, 139-59. [3] Yuan, W., Dong, H., Li, C.M., Cui, X., Yu, L., Lu, Z. and Zhou, Q. (2007) Langmuir, 23, 13046-52.

The exponential development of biosensors as powerful analytical tools in the last four decades mainly relies on the high sensitivity and selectivity offered when detecting the target analyte. The transducer and the biological receptor are the bases of the biosensor development. Nevertheless, the bioreceptor immobilisation is also important, playing a key role in the retention of the biological activity, and thus affecting the sensitivity. Parameters such as shelf-life and surface regeneration also depend on the biomolecule immobilisation. Researchers are focusing their efforts towards random and oriented immobilisation procedures. Adsorption, entrapment, cross-linking and electrostatic interactions provide randomly immobilised biomolecules, sometimes partially hindering their biological activity. Covalent binding and affinity interactions may enable oriented biomolecule immobilisations, providing controlled, reproducible and highly active modified surfaces. This paper reviews the main immobilisation strategies used in the biosensors development, putting special emphasis on our contribution to mild and oriented immobilisation techniques.

Protein-based voltammetric biosensors are sensors based on the electric communication between proteins and electrodes. Recently, more and more nanomaterials are utilized to assist the fabrication of such kind of biosensors. In this review, we mainly detail the biosensors constructed with different kinds of nanomaterials depending on their categories in the past two years.

Optical reagentless biosensors are one of the most promising alternatives for producing selective, sensitive and autonomous sensors for real life applications. These devices are based on the efficient use of the spectroscopic properties of bioreagents, mainly proteins, as transducers; avoiding in this way the use of chemical colorant/fluorophores which usually limit sensors performance. In this paper a brief state of the art of the bioreagents being used in biosensors as well as recent alternatives are discussed. The advantages of flavoenzymes and hemeproteins as the basis for reagentless biosensors are particularly stressed.

We developed an amperometric propionate sensor using comprised of two recombinant enzymes, propionate coenzyme A CoA transferase from Clostridium propionicum and short-chain acyl-CoA oxidase from Arabidopsis thaliana. Response current increased linearly with increase in propionate concentration from 10 μM to 100 μM. The detection limit was 10 μM propionate.

A new approach toward the development of advanced immunosensors based on chemically functionalized coreshell- shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of α-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.

This work describes the investigations of electrochemical interactions of naphthalene acetic acid (NAA) with stabilized lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated auxin-binding protein 1 receptor for the development of a biosensor for the rapid detection of this compound in fruits. NAA was injected into the flowing streams of a carrier electrolyte solution, the flow of the electrolyte solution stops and an ion current transient was obtained; the magnitude of the signal was correlated to NAA concentration, which could be determined at the micromolar level. NAA preconcentrates at the lipid membrane surface which causes dynamic alterations of the electrostatic fields and phase structure of membranes. The response times were ca. 5 min and naphthalene acetic acid was determined at concentration levels of μM. The effect of potent interferences included a wide range of compounds. The results showed no interferences from these compounds in concentration levels usually found in real samples. The method was applied for the determination of NAA in fruits and the reproducibility of the method was checked in about 100 samples. A quantitative method for the detection of NAA in fruits that can be complimentary to HPLC methods is provided in the present paper. These lipid films can be used as portable sensors for the rapid detection of NAA in fruits by non-skilled personnel.

Biosensors using cholinesterases as the biorecognition component have been used to assay organophosphates and carbamates for a long time. In this review, some strategies convenient for biosensor construction are presented. Solutions for cholinesterase immobilization and output signal monitoring are presented as the basic presumptions for successful biosensor construction.

We report on a new type of artificial receptor formed by hybridization of two DNA aptamers for human thrombin (aptabody). This aptasensor based on multiwalled carbon nanotubes allowed us to detect thrombin with detection limit of 0.3 nM, which was 3 times better in comparison with conventional aptamer.

The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.

Circular dichroism reports that glucagon in solution becomes increasingly helical with the addition of fluoroalcohol (which also decreases solution pH), and with changes in pH and ionic strength. Given the variability of structure observed, these data indicate that care must be taken when comparing results obtained under different solution conditions.

Heat stress on structure and ligand binding of β-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of β-LG is reversible up to 75°C then it becomes irreversible due to aggregation of β-LG. Formation of aggregated β-LG is completed at 95°C. Circular dichroism results indicate that formation of aggregated β-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the β↔α structure. In the presence of retinol, the β-helix content of the secondary structure of heat-treated β-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated β-LG at 95°C can still bind retinol. The refolding of the tertiary structure of β-LG heat-denatured at 95°C may recreate a retinol binding site. Surprisingly, the affinity of the new site for retinol is higher than that of native β-LG; however, the apparent molar ratio is lower than one. The binding properties of β-LG for terpenoids have been measured after its heat treatment at 20, 75 and 95°C. The intensity of tryptophan emission at 330 nm was changed only in the case of the interaction with β-ionone. Other ligands probably cannot bind to β-LG or they bind in a binding site far from the tryptophan residues, hence not affecting its fluorescence.

G-protein coupled receptors (GPCRs) are involved in various physiological processes. Therefore, classification of amine type GPCRs is important for proper understanding of their functions. Though some effective methods have been developed, it still remains unknown how many and which features are essential for this task. Empirical studies show that feature selection might address this problem and provide us with some biologically useful knowledge. In this paper, a feature selection technique is introduced to identify those relevant features of proteins which are potentially important for the prediction of amine type GPCRs. The selected features are finally accepted to characterize proteins in a more compact form. High prediction accuracy is observed on two data sets with different sequence similarity by 5-fold cross-validation test. The comparison with a previous method demonstrates the efficiency and effectiveness of the proposed method.

This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.

Advanced glycation end products (AGEs)-their receptor (RAGE) axis plays a central role in the pathogenesis of diabetic microangiopathy. Since the pathophysiological crosstalk between the AGEs-RAGE system and angiotensin II has also been associated with diabetic microangiopathy, we examined here whether and how telmisartan, a unique angiotensin II type 1 receptor blocker (ARB) with peroxisome proliferator-activated receptor-γ (PPAR-γ)-modulating activity, could inhibit the AGEs-elicited endothelial cell injury by suppressing RAGE expression in vitro. Telmisartan suppressed RAGE expression at both mRNA and protein levels in human cultured microvascular endothelial cells (ECs), which were prevented by GW9662, an inhibitor of PPAR-γ. Further, telmisartan was found to inhibit up-regulation of mRNA levels for monocyte chemoattractant protein-1, intercellular adhesion molecule-1 and vascular endothelial growth factor in AGEsexposed ECs. These results suggest that telmisartan inhibits the AGEs-elicited EC injury by down-regulating RAGE expression via PPAR-γ activation. Our present study provides a unique beneficial aspect of telmisartan. Specifically, it could work as an anti-inflammatory agent against AGEs via PPAR-γ activation and may play a protective role against diabetic microangiopathy.

A recombinant human parathyroid hormone (rhPTH) fragment (Gly1-Gln26-rhPTH(1-34)) which contains two amino acids substitutions (Gly1 and Gln26) was acquired through Escherichia coli expression system using a soluble fusion protein strategy. The soluble fusion protein MBP-Gly1-Gln26-rhPTH(1-34) was harvested after purification by Phenyl-Sepharose F.F and Q-Sepharose F.F chromatographies. Following tobacco etch virus (TEV) protease cleavage and further purification by SP-Sepharose F.F chromatography, 30.8 mg/L Gly1-Gln26-rhPTH(1-34) without tag was obtained with high purity up to 99%. Cyclic AMP (cAMP) stimulation assay suggested that Gly1-Gln26-rhPTH(1-34) could increase the biological activity by up to 13.89% and 6.34%. After daily subcutaneous injection (for 13 weeks) of 5, 10 and 20 μg of Gly1-Gln26-rhPTH(1-34)/1000g body weight, the mean Bone Material Density (BMD) of ovariectomized (OVXed) rats increased to 7.95-30.54% and 1.98-23.32%, compared to control-vehicle group (OVX, P<0.001) and sham- operated group (SHAM, P<0.01), respectively.

To develop antimicrobial peptides having higher bacterial selectivity than a novel antimicrobial peptide P18, we synthesized several analogues. The P18 analogues are designed by movement of the N-terminal Trp2 residue in P18 (P18-W6, P18-W8 and P18-W15) and the substitution of the central Pro9 residue with D-Pro or Nala (P18-Nala9 and P18- D-Pro9). These analogues retained potent antibacterial activity but displayed less hemolytic activity than P18. From the viewpoint of their therapeutic index, P18 analogues had approximate 3- to 7-fold higher bacterial selectivity compared to P18. The analogues preferentially bind to bacterial membrane-mimicking negatively charged liposomes as well as does P18. Their high specificity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Furthermore, P18-W6, P18-W8 and P18-Nala9 induced a significant inhibition in NO production from LPS-stimulated macrophage RAW264.7 cells, as well as P18. This result suggests that these peptides appear to have promising therapeutic potential for future development as a novel anti-inflammatory agent as well as antimicrobial agent.