Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) x10(12)/cm2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 x 10(12)/cm2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.     

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.     

Glutathione amide and its perthiol in anaerobic sulfur bacteria.

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.     

Synthesis of a near-infrared fluorescent probe and its application in imaging of MCF-7 cells

IR-789, a novel near-infrared fluorescent probe, was designed, synthesized, and applied to living cells. The probe exhibited better response fluorescence characteristics than the only FDA-approved agent, indocyanine green. Cell experiments showed that the probe had high affinity and without apparent cytotoxicity. Fluorescent image experiments in living MCF-7 cells (human breast adenocarcinoma cell line) further demonstrated the potential applications of the probe in biological systems. The probe effectively prevented the influence of autofluorescence and native cellular species in biological systems. It also exhibited high sensitivity, good photostability, and excellent cell membrane permeability.     

A FRET-based method to study protein thiol oxidation in histological preparations

Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique-which can be used in association with standard immunological staining procedures-to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool for providing further understanding of oxidative imbalance, a phenomenon common to many diseases.     

Molecular cloning of a bovine cathepsin.

A cDNA clone for a thiol endoproteinase has been isolated from a bovine heart cDNA library by using a mixture of 32 synthetic oligonucleotides as a hybridization probe. The inserted region is 672 base pairs in length. It contains a sequence encoding the C-terminal region of a protein that is homologous to rat liver cathepsins B and H and to plant thiol proteinases. In addition, it contains the sequence of 442 bases corresponding to the 3' untranslated region of the mRNA. The inserted region was used as a specific probe in RNA transfer analysis; the size of the mRNA encoding the thiol endoproteinase is estimated to be approx. 1.7 kilobases. Thus, the maximum size of the encoded protein is about 350-400 amino acids.     

Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase

The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet yield of these probe molecules. Consistent with this, the greatest effect is seen with erythrosin isothiocyanate and the least effect with fluorescein isothiocyanate. These reductions of ATPase activity associated with illumination are also associated with an aggregation of the protein molecules. This is indicated by laser flash photolysis measurements and also by polyacrylamide gel electrophoresis. A reduction in the number of thiol groups present on the ATPase molecule parallels the reduction of enzyme activity and changes in the protein mobility. The results are discussed in relation to the use of these probe molecules to study biological systems and also in terms of oxidative processes which may affect protein function in vivo.     

Relative reactivities of N-chloramines and hypochlorous acid with human plasma constituents

Hypochlorous acid (HOCl), the major strong oxidant produced by the phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Since different N-chloramines have different stabilities and reactivities depending on their structures, we investigated the relative reactivities of three model N-chloramines and HOCl with human plasma constituents. TheN-chloramines studied were N(alpha)-acetyl-lysine chloramine (LysCA, a model of protein-associated N-chloramines), taurine chloramine (TaurCA, the primary N-chloramine produced by activated neutrophils), and monochloramine (MonoCA, a lipophilic N-chloramine). Addition of these chlorine species (100--1000 microM each) to plasma resulted in rapid loss of thiols, with the extent of thiol oxidation decreasing in the order TaurCA = LysCA > MonoCA = HOCl. The single reduced thiol of albumin was the major target. Loss of plasma ascorbate also occurred, with the extent decreasing in the order HOCl > LysCA > TaurCA > MonoCA. Experiments comparing equimolar albumin thiols and ascorbate showed that while HOCl caused equivalent loss of thiols and ascorbate, theN-chloramines reacted preferentially with thiols. The chlorine species also inactivated alpha(1)-antiproteinase, implicating oxidation of methionine residues, and ascorbate provided variable protection depending on the chlorine species involved. Together, our data indicate that in biological fluids N-chloramines react more readily with protein thiols than with methionine residues or ascorbate, and thus may cause biologically relevant, selective loss of thiol groups.     

Biochemical characterization of S-nitrosohemoglobin. Mechanisms underlying synthesis, no release, and biological activity.

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanisms responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives have been synthesized and characterized. HbO(2) can be nitrosated at up to three distinct residues, one in the alpha-globin chain and two in the beta-chain. A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-dependent mechanism involving nucleophilic attack at the nitrosated thiol functionality of SNO-Hb; in the case of glutathione, this process was associated with the formation of a mixed disulfide. In contrast, multinitrosated hemoglobin species released NO and relaxed vascular smooth muscle by a thiol-independent mechanism. HbO(2) scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative intermediate; the presence of a low molecular weight thiol is a prerequisite for this process. Such observations have important implications for the generation, metabolic fate, and biological activity of S-nitrosothiols.     

Zinc protects HeLa-tat cells against free radical cytotoxicity induced by glucose

In the present study, we investigated the protective effect of zinc on the glucose-induced cytotoxicity in HeLa wild and HeLa-tat cells (30 and 20 mmol/l glucose, respectively). HeLa cells transfected with the protein Tat exhibit a lower antioxidant defense system. Incubation of HeLa wild and HeLa-tat cells with high glucose levels led to a rapid increase in generation of reactive oxygen species (ROS). As expected in the presence of high glucose concentrations, the viability was reduced for both cell lines. The redox status essentially regulated by thiol groups may play an important role in the apoptotic process. Thus, we developed a new method using the p-nitrophenyl disulfide to measure cytosolic thiol groups in intact cells. Cellular zinc was measured using inductively coupled plasma mass spectrometry. Intracellular thiol groups and intracellular zinc concentrations were significantly lower in HeLa cells cultured in hyperglycemic conditions, and their concentrations were significantly lower in HeLa-tat cells than in HeLa wild cells. However, the generation of ROS and the induction of apoptosis by a glucose specific mechanism were prevented by zinc (50 micromol/l) and the intracellular thiol groups and zinc concentrations significantly increased in both cell lines to become similar to the initial values. These results suggest that the glucose oxidation and its subsequent effects on the cells can be prevented by a biological antioxidant such as zinc.     

Thiol peptide hydrolases from animal tissues, their structure and function

Data on properties, structure and biological functions of a variety of thiol (cysteine) peptide hydrolases from animal tissues have been summarized. This large group of diverse intracellular enzymes involves both endo- and exopeptidases. Best studied are lysosomal thiol peptide hydrolases: cathepsins B, H and L, the primary structure of which is deciphered. They present a family of homologous proteins, structurally similar to papain. Ca2+-dependent neutral proteinases is another family of related proteins. The biological functions of various thiol peptide hydrolases are considered: their participation in protein turnover, post-translational processing, regulation of unidirectional biological processes and metabolic refolding. Data on endogenous inhibitors of thiol peptide hydrolases and on regulation of enzymic activity are presented.     

Comparative kinetics of thiol oxidation in two distinct free-radical generating systems: SIN-1 versus AAPH

Abstract To study oxidative stress in biological systems, chemical compounds capable of producing free radicals have been widely used. Here, we compared two free-radical generators, 3-morpholinosydnonimine (SIN-1) and 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH), by measuring the thiol oxidation kinetics of various thiols. We found that SIN-1 is >?30 times potent in causing thiol oxidation than AAPH. Kinetic simulations revealed that in the SIN-1 system (0.1 mM), superoxide, nitrogen dioxide and carbonate radicals are the major reactive species which, in combination, induce ～50% of thiol molecules to undergo one-electron oxidation, thereby forming the thiyl radical which propagates further thiol oxidation by direct coupling with thiolates. Similarly, the alkyl peroxyl radical derived from AAPH (3 mM) initiates comparable extent of one-electron oxidation and formation of the thiyl radical. In conclusion, our study provides experimental and theoretical evidence that SIN-1 is mainly an one-electron oxidizing agent that can be functionally mimicked by AAPH.     

A fast‐responsive fluorescent probe for sulfite and its bioimaging

A turn‐on fluorescent probe Coumarin‐SO2 based on a nucleophilic addition reaction was developed for the rapid detection of SO₃^2– in aqueous media. The probe Coumarin‐SO2 displays excellent water solubility, fast response, highly sensitivity and highly selectivity over other biological related species. More importantly, living cell imaging experiments indicate the feasibility of using the probe for the detection of SO₃^2– in biological systems. Copyright © 2015 John Wiley & Sons, Ltd.     

Mitochondrial dysfunction in cardiac ischemia–reperfusion injury: ROS from complex I,without inhibition

A key pathologic event in cardiac ischemia reperfusion (I–R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I–R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1α sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I–R injury are discussed.     

Mitochondrial dysfunction in cardiac ischemia-reperfusion injury: ROS from complex I, without inhibition

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.     

A novel reversible thiol-specific spin label: papain active site labeling and inhibition

A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5–7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.     

Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols.

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.     

DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection

The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.     

A simple rhodamine hydrazide‐based turn‐on fluorescent probe for HOCl detection

Hypochlorous acid (HOCl) plays a crucial role in daily life and mediates a variety of physiological processes, however, abnormal levels of HOCl have been associated with numerous human diseases. It is therefore of significant interest to establish a simple, selective, rapid and sensitive fluorogenic method for the detection of HOCl in environmental and biological samples. A hydrazide‐containing fluorescent probe based on a rhodamine scaffold was facilely developed that could selectively detect HOCl over other biologically relevant reactive oxygen species, reactive nitrogen species and most common metal ions in vitro. Via an irreversible oxidation–hydrolysis mechanism, and upon HOCl‐triggered opening of the intramolecular spirocyclic ring during detection, the rhodamine hydrazide‐based probe exhibited large fluorescence enhancement in the emission spectra with a fast response, low detection limit and comparatively wide pH detection range in aqueous media. The probe was further successfully applied to monitoring trace HOCl in tap water and imaging both exogenous and endogenous HOCl within living cells. It is anticipated that this simple and useful probe might be an efficient tool with which to facilitate more HOCl‐related chemical and biological research. Copyright © 2015 John Wiley & Sons, Ltd.