Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Chronopotentiometric sensing of native, oligomeric, denatured and aggregated serum albumin at charged surfaces.

In protein analysis, fast techniques applicable for preliminary tests of the protein structural changes are sought. We show that using constant current chronopotentiometric stripping peak H, small amounts of oligomeric, denatured and aggregated bovine serum albumin (BSA) can be easily distinguished from native form. Different behavior of native, denatured, and aggregated BSA could be explained by combination of their different adsorption at charged surface and accessibility of electroactive amino acid residues. Ability to discriminate between individual forms allows to use chronopotentiometric stripping for study of processes responsible for structural changes, such as freezing treatment.

AGR2-AGR3 hetero-oligomeric complexes: Identification and characterization.

In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.

Bovine Serum Albumin Catalysed Hydrogen and Deuterium Evolution at Mercury Electrodes.

The hydrogen evolution reaction (HER), catalysed by proteins at mercury electrodes and reflected in chronopotentiometric stripping peak H, provides a label-free and reagentless analytical technique that is sensitive to protein structure. Here we show how the kinetic isotope effect affected the HER catalysed by the protein bovine serum albumin (BSA). We found that the deuteron bond, which is stronger than that of a proton, contributed to less effective transport of deuterons mediated by BSA at the Hg|D2 O interface, and enhanced structural stability of the surface-attached native BSA in D2 O solution. A structural transition was also observed in the surface-attached urea-denatured BSA, and is probably due to the destabilisation of some secondary structural remnants retained by the 17 SS-bonds. Because the catalytically active groups involved in proton or deuteron transfer in native proteins are often exposed towards solutions and their protons exchange almost instantly, no signs of H/D exchange were observed in native BSA using peak H under the given conditions.

Influence of Protein Modification and Glycosylation in the Catalytic Hydrogen Evolution Reaction of Avidin and Neutravidin: An Electrochemical Analysis.

To investigate glycans' influence on the behavior of glycoproteins on charged surfaces, avidin and its nonglycosylated and neutralized version neutravidin were studied by label-free chronopotentiometric stripping (CPS) analysis and alternating current voltammetry combined with a mercury electrode. Despite neutravidin's and avidin's similar size and structure, their CPS responses differed due to the different amounts of catalytically active free amino groups of lysine and arginine residues. Acetylation of the proteins resulted in the suppression of their CPS responses by almost four times for avidin and by about 50 % for neutravidin, respectively. On the other hand, the presence of glycans in the acetylated avidin induced about 30 % higher chronopotentiometric response compared to the acetylated neutravidin. We suggest that the presence, size and composition of the glycans influenced the CPS signal due to differences in the orientation at a charged surface. The obtained results can be utilized in glycoprotein research.

Distinguishing the glycan isomers 2,3-sialyllactose and 2,6-sialyllactose by voltammetry after modification with osmium(VI) complexes.

Altered glycosylation is a universal feature of cancer cells and certain glycans are well-known markers of tumor progression. In this work we studied two glycan isomers, 2,3-sialyllactose (3-SL) and 2,6-sialyllactose (6-SL), frequently appearing in glycoproteins connected with cancer. A combination of square wave voltammetry and glycan modification with osmium(VI) N,N,N',N'-tetramethylethylenediamine (Os(VI)tem) allowed to distinguish between these regioisomers, since the 6-SL molecule can bind three Os(VI), while the 3-SL only two Os(VI) moieties, as experiments using capillary electrophoresis, inductively coupled plasma mass spectrometry and thin layer chromatography showed. A similar pattern of Os(VI)-modification was found for isomers of sialyl-N-acetyllactosamine and sialylgalactose. Covalent adducts of Os(VI)tem with glycans yielded three reduction voltammetric peaks. The ratio of peak I/peak II heights depends on the content of individual regioisomer in the sample. Our proposed approach allows the determination of isomer percentage representation in the mixture after one voltammogram recording. These results show a new appropriate method for the discrimination of glycan isomers containing terminal sialic acid important for distinguishing between cancerous and non-cancerous origin of biomarkers.

Label-free chronopotentiometric glycoprofiling of prostate specific antigen using sialic acid recognizing lectins.

In recent decades, it has become clear that most of human proteins are glycosylated and that protein glycosylation plays an important role in health and diseases. At present, simple, fast and inexpensive methods are sought for clinical applications and particularly for improved diagnostics of various diseases, including cancer. We propose a label- and reagent-free electrochemical method based on chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode for the detection of interaction of sialylated protein biomarker a prostate specific antigen (PSA) with two important lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). Incubation of PSA-modified electrode with specific SNA lectin resulted in an increase of CPS peak H of the complex as compared to this peak of individual PSA. By adjusting polarization current and temperature, PSA-MAA interaction can be either eliminated or distinguished from the more abundant PSA-SNA complex. CPS data were in a good agreement with the data obtained by complementary methods, namely surface plasmon resonance and fluorescent lectin microarray. It can be anticipated that CPS will find application in glycomics and proteomics.

Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer.

Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

Electrochemical sensing of tumor suppressor protein p53-deoxyribonucleic acid complex stability at an electrified interface.

Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA-protein complex nanolayers. Rapid potential changes at high negative current intensities (Istr) in CPS are utilized in the analysis of DNA-protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD-DNA complex. By adjusting Istr and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD-DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10-20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low I(str) intensities are deleterious to the p53CD-DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.

Enzymatic activity and catalytic hydrogen evolution in reduced and oxidized urease at mercury surfaces.

It was originally shown [10] that urease retains its enzymatic activity when adsorbed at bare mercury and solid amalgam surfaces. However the opinion later prevailed that, when adsorbed at bare metal electrodes, proteins are irreversibly denatured. Here we confirm that urease is enzymatically active at a bare solid amalgam surface as found by Santhanam et al., and we show that this enzyme is equally active at a thiol-modified amalgam surface. We also show that it is the reduced form of urease, which is enzymatically active at Hg surfaces. Oxidation of the protein, resulting in formation of disulfide bonds, strongly decreases the enzyme activity. Using constant current chronopotentiometric stripping (CPS) we show that the exposure of surface-attached urease to negative potentials results in the protein unfolding. The extent of the unfolding depends upon the amount of time for which the protein is exposed to negative potentials, and at very short times this unfolding can be avoided. At thiol-modified Hg surfaces the protein is less vulnerable to the effects of the electric field. We conclude that the loss of enzymatic activity, resulting from a 10 min exposure of the protein to -0.58 V, is not due to reduction of the disulfide bonds as suggested by Santhanam et al. This loss is probably a result of protein reorientation, due to reduction of the Hg-S bonds (formed by accessible cysteines), followed by prolonged electric field effect on the surface-attached protein.

Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes.

In an attempt to develop a label-free electrochemical method for detection of changes in protein structures based on oxidizability of tyrosine and tryptophan residues we tested different types of carbon electrodes. We found that using edge plane pyrolytic graphite electrode (EPGE) we can discriminate between native and denatured forms of human serum albumin (HSA) and of other proteins, such as bovine and chicken serum albumin, aldolase and concanavalin. Treatment of natively unfolded α-synuclein with 8 M urea resulted only in a small change in the tyrosine oxidation peak, in a good agreement with absence of highly ordered structure in this protein. Using square wave voltammetry with EPGE we were able to follow the course of HSA denaturation at different urea concentrations. The electrochemical denaturation curve agreed reasonably well with that based on intrinsic fluorescence of tyrosine and tryptophan. It can be expected that the electrochemical method will be applicable to a large number of proteins and may become useful in biomedicine and proteomics.

Simple protein structure-sensitive chronopotentiometric analysis with dithiothreitol-modified Hg electrodes.

We have shown that proteins produce at bare mercury electrodes a well-developed chronopotentiometric peak H. At sufficiently high current densities and low ionic strengths, this peak is sensitive to changes in protein structures. At higher ionic strengths this sensitivity can be lost but it can be restored, when instead of bare, thiol-modified Hg electrodes are used. Here we studied properties of the dithiothreitol (DTT) layer at the hanging mercury drop electrode and showed that at low concentrations (5 µM-200 µM) the DTT is adsorbed as a dithiol with both -SH groups attached to the surface. At higher DTT concentrations than 1mM, a densely packed pinhole-free layer is formed with the DTT molecules bound to the electrode surface by a single -SH group, oriented perpendicularly to the surface. We found that, if a sufficiently high DTT concentration is used, preparation of the DTT-modified Hg electrodes can be omitted and proteins can be co-adsorbed with DTT on liquid Hg or solid amalgam electrodes without the loss of sensitivity for changes in protein structures. The newly observed properties of the DTT self assembled monolayers (SAMs) at Hg electrodes appear important for designing new types of solid amalgam electrode arrays for electrochemical analysis of proteins.

Electrocatalytic monitoring of metal binding and mutation-induced conformational changes in p53 at picomole level.

We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.

Protein structure-sensitive electrocatalysis at dithiothreitol-modified electrodes.

Dithiothreitol (DTT)-mercury and DTT-solid amalgam electrodes are proposed for protein microanalysis by means of constant current chronopotentiometric stripping (CPS). At the DTT-modified hanging mercury drop electrode (DTT-HMDE), proteins at nanomolar concentrations produce the CPS peak H, which is due to the protein catalyzed hydrogen evolution. Self-assembled monolayers (SAMs) of DTT at the electrode surface protected surface-attached proteins from the electric field-driven denaturation, but did not interfere with the electrocatalysis. Using CPS peak H, native and denatured forms of bovine serum albumin (BSA) and of other proteins were easily distinguished. On the other hand, in usual slow scan voltammetry (scan rates between 50 mV/s and 1 V/s), the adsorbed BSA behaved as fully or partially denatured. BSA-modified DTT-HMDE was exposed to different potentials, E(B) for 60 s, followed by CPS measurement. Three E(B) regions were observed, in which either BSA remained native (A, -0.1 to -0.3 V), was denatured (B, -0.35 to -1.4 V), or underwent desorption (C, at potentials more negative than -1.4 V). At potentials more positive than the reduction potential of the DTT Hg-S bond (approximately -0.65 V against Ag|AgCl|3 M KCl), the densely packed DTT SAM was impermeable to [Ru(NH(3))(6)](3+). At more negative potentials, the DTT SAM was disturbed, but under conditions of CPS (with very fast potential changes), this SAM still protected the protein from surface-induced denaturation. Thiol-modified Hg electrodes in combination with CPS represent a new tool for protein analysis in biomedicine and proteomics.

Base-modified DNA labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes: construction by polymerase incorporation of modified nucleoside triphosphates, electrochemical and luminescent properties, and applications.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.

Osmium tetroxide, 2,2'-bipyridine: electroactive marker for probing accessibility of tryptophan residues in proteins.

A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.