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The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables; 6 of them, including yeast extract, glucose, peptone and cysteine; temperature and agitation rate had a positive significant effect on GSH production with a maximum production of 192 mg/L. The impact of addition time of cysteine was investigated in 19 experiments during the growth time course (0-36 h), the best addition time was 8h post-inoculation producing 235 mg/L of GSH. The most significant variables were further explored at 5-levels using Central Composite Rotatable Design (CCRD), giving a maximum production of GSH (552 mg/L). Using baffled flasks, the GSH was increased to 730 mg/L, i.e 1.32-folds increment than obtained using CCRD. The two rate-limiting genes of GSH biosynthesis “γ-glutamyl cysteine synthetase (gsh1) and GSH-synthetase (gsh2) were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with gsh1 and gsh2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80 kDa and 35 kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-folds upon demetallization confirming its metalloproteinic identity. The enzyme was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.

Purpose To investigate the effect of geniposide on the biosynthesis of insulin and the expression protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductin 1 (ERO1) in the presence of low (5 mM) and high (25 mM) glucose in pancreatic β cells. Methods The content of insulin was measured by ELISA, the number of SH groups was determined with the classical chromogenic reagent, 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB; also known as Ellman's reagent), the expressions of PDI and ERO1 were analyzed by Western blot. Results Geniposide played contrary roles on the accumulation of H2O2, the ratio of GSH/GSSG and the thiol-disulfide balance in the presence of low (5 mM) and high (25 mM) glucose in rat pancreatic INS-1 cells. Geniposide also regulated the protein levels of protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductin1 (ERO1), the two key enzymes for the production of H2O2 during the biosynthesis of insulin in INS-1 cells. Conclusion Geniposide affects glucose-stimulated insulin secretion by modulating the thiol-disulfide balance that is controlled by the redox signaling in pancreatic β cells.

Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.

Researchers are motivated to build effective Intrusion Detection Systems because of the implications of malicious actions in computing, communication, and cyber-physical systems (IDSs). In order to develop signature-based intrusion detection techniques that are suitable for use in cyber-physical environments, state-of-the-art supervised learning algorithms are devised. The main contribution of this research is the introduction of a signature-based intrusion detection model that is based on a hybrid Decision Table and Naive Bayes technique. In addition, the contribution of the suggested method is evaluated by comparing it to the existing literature in the field. In the preprocessing stage, Multi-Objective Evolutionary Feature Selection (MOEFS) feature selection has been used to select only five attack features from the recent CICIDS017 dataset. Keeping in view the class imbalance nature of CICIDS2017 dataset, adequate attack samples has been selected with more weightage to the attack classes having a smaller number of instances in the dataset. A hybrid of Decision Table and Naive Bayes models were combined to train and detect intrusions. Detection of botnets, port scans, Denial of Service (DoS)/Distributed Denial of Service (DDoS) attacks, such as Golden-Eye, Hulk, Slow httptest, slowloris, Heartbleed, Brute Force attacks, such as Patator (FTP), Patator (SSH), and Web attacks such as Infiltration, Web Brute Force, SQL Injection, and XSS, are all successfully detected by the proposed hybrid detection model. The proposed approach shows an accuracy of 96.8% using five features of CICIDS2017, which is higher than the accuracy of methods discussed in the literatures.

This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman′s reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.

Food colorants are widely used by humans in food production and preparation, however, their potential toxicity requires an in-depth analysis. In this study, five out of 15 commercial food colorants, namely, lutein, betanin, caramel, crocin and chlorophyll, significantly inhibited wild type selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1) in vitro. The hyperactive Sec498 residue of TrxR1 was targeted by those five colorants, which was confirmed by the site-directed mutagenesis of TrxR1. Furthermore, two colorants, chlorophyll and betanin, triggered the oligomerization of TrxR1. A chlorophyll-derived compound, chlorophyllin, irreversibly inhibited the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) reducing activity of TrxR1 with Kinact = 6.96 × 10−3 ± 0.49 × 10−3 µM−1 min−1. Moreover, chlorophyllin reduced the cellular TrxR activity, leading to reactive oxygen species (ROS) accumulation and, subsequently, promoting cancer cell death. In conclusion, this study might contribute to understand the food safety of commercial colorants and provide chemotherapeutic compounds by targeting TrxR1.

Substrates based on the metal-polymer nanocomposites 2,3-dichloro-p-xylylene-silver (PCPX-Ag) that realize the effect of surface-enhanced Raman scattering (SERS) were developed. To obtain nanocomposites, the vapor-phase polymerization method was used (VDP), which makes it possible to control the nanocomposite microstructure. In the process of self-assembly during VDP, nanocomposite films with inclusions of silver particles were formed on the polycore substrates. Silver content varied from 2.5 to 16% vol. The possibility of using such substrates for the detection of low-molecular substances, for example 5,5′-dithiobis- (2-nitrobenzoic acid) (DTNB) analyte, by the SERS method with an enhancement factor of up to 104, was demonstrated. The dependence of the SERS spectra enhancement on the microstructure of the nanocomposite and the silver content was determined. The optical and morphological properties of nanocomposites were also investigated and their dependence on the silver content was shown. It has been demonstrated that the nanocomposite is SERS selective since when working with complex solutions in the presence of high molecular weight substances, signal enhancement was only observed for low molecular weight substances.

Alzheimer’s disease (AD) is a genetically complex disease for which roughly 30 genes have been identified via genome-wide association studies. We attempted to identify rare variants (minor allele frequency −6, using the burden test or the SKAT statistic. The genomic region around the dystobrevin beta (DTNB) gene was identified with the burden test and replicated in case/control samples from the ADSP study (pmeta = 4.74×10−8). SKAT analysis revealed region-based association around the discs large homolog 2 (DLG2) gene and replicated in case/control samples from the ADSP study (pmeta =1×10−6). Here, in a region-based GSAS of AD we identified two novel AD genes, DLG2 and DTNB, based on association with rare variants.

We characterized the metal tolerance of recombinant strains harboring metallothionein from the freshwater crab Sinopotamon henanense (ShMT) in vivo and metal binding properties of ShMT purified in vitro. The recombinant strains harboring ShMT were exposed to 0.1 mM Cd2+, 0.3 mM Cu2+, 0.5 mM Pb2+, and 0.8 mM Zn2+. The growth curves and spot assays of recombinant strains and the contents of heavy metal ions were analysed in the media supplemented with above metal ions provided to recombinant E. coli synthesis. The structural characteristics of the Cd-, Cu-, Pb-, and Zn-ShMT were determined through ultraviolet spectroscopy (UV-vis), circular dichroism (CD), and isothermal titration calorimetry (ITC). The in vivo results showed that, compared to control strains, recombinant strains tolerated Cd2+, Cu2+, Pb2+, and Zn2+. Furthermore, the contents of Cd2+ and Pb2+ in media decreased substantially. In vitro and the Cd-ShMT had a higher degree of folding compactness in solution. 5,5′-Dithiobis-(2-nitrobenzoic) acid (DTNB) reaction and ITC results demonstrated that ShMT yielded Cd6-, Cu7-, and Pb6-ShMT. The binding stability order was Cu-ShMT > Cd-ShMT > Pb-ShMT > Zn-ShMT. Overall, ShMT is a canonical crustacean MT and is defined as a Cd-specific MT isoform that functions mainly in a detoxifying Cd2+ and Pb2+ and in regulating Zn2+ homeostasis in S. henanense. This research on the metal binding properties of ShMT provides a better understanding of the physiological function of ShMT reducing heavy metal bioavailability and by regulating essential trace metals.

Background/aims Magnesium, whose supplementation provides beneficial effects against oxidative stress-related conditions, has been here used to possibly protect Band 3 protein anion exchange capability and underlying signaling in an in vitro model of oxidative stress. Methods Whole blood samples pre-exposed to 10 mM MgCl2, were treated for 30 min with H2O2 (300 µM, 600 µM and 1 mM) chosen as oxidant molecule. In a separate protocol, NEM (0.5,1 and 2 mM), a phosphatase inhibitor and thiol-alkilant agent, has been also applied. The rate constant for SO4= uptake, accounting for Band 3 protein anion exchange capability, has been measured by a turbidimetric method, while intracellular reduced glutathione (GSH) levels and membrane -SH groups mostly belonging to Band 3 protein were spectrophotometrically quantified after reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid). Expression levels of Band 3 protein, phosporylated Tyrosine (P-Tyr) and tyrosine kinase (Syk) involved in signaling have been also measured. Results Our results show that Mg2+ prevented the reduction in the rate constant for SO4= uptake on H2O2-treated erythrocytes, not involving GSH levels and membrane -SH groups, unlike NEM, remaining both P-Tyr and Syk expression levels high. Conclusion Hence, i) the measurement of the rate constant for SO4= uptake is a useful tool to evaluate Mg2+ protective effect; ii) the use of two different oxidant molecules shed light on Mg2+ effect which seems not to modulate phosphorylative pathways but would putatively stabilize membrane organization; iii) the use of Mg2+ in food supplementation can be reasonably supported to protect erythrocytes homeostasis in case of oxidative stress-related diseases.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH₂CH₂SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

Nicotine causes neurotoxic effects because it quickly penetrates the blood–brain barrier after entering the human body. Acetylcholinesterase (AChE) is a key enzyme in the central and peripheral nervous system associated with neurotoxicity. In this study, a spectroscopic method and computer simulation were applied to explore the mode of interaction between AChE and enantiomers of nicotine (S/R-nicotine). Fluorescence spectroscopy showed that the quenching mechanism of endogenous fluorescence of AChE by S/R-nicotine was static, as confirmed by the time-resolved steady-state fluorescence. The binding strength of both nicotine to AChE was weak (S-AChE: Ka = 80.06 L mol−1, R-AChE: Ka = 173.75 L mol−1). The main driving forces of S-AChE system interaction process were van der Waals force and hydrogen bonding, whereas that of R-AChE system was electrostatic force. Computer simulations showed that there were other important forces involved. S/R-Nicotine had a major binding site on AChE, and molecular docking showed that they bound mainly to the cavities enclosed by the active sites (ES, PAS, OH, AACS, and AP) in the protein. UV-vis spectroscopy and 3D spectroscopy indicated that nicotine significantly affected the microenvironment of Trp amino acids in AChE. The CD spectra indicated that S-nicotine increased the α-helical structure of AChE, but the overall conformation did not change significantly. By contrast, R-nicotine significantly changed the secondary structure of AChE. 5,5′-Dithiobis-2-nitrobenzoic acid (DTNB) method indicated that S and R nicotine produced different degrees of inhibition on the catalytic activity of AChE. Both experimental methods and computer simulations showed that R-nicotine had a significantly higher effect on AChE than S-nicotine. This research comprehensively and systematically analyzed the mode of interaction between nicotine and AChE for neurotoxicity assessment.

The redox potential of goat serum was assessed by different spectrophotometric assays. Among them, three methods are commonly applied for the evaluation of the oxidative (reactive oxygen metabolites, ROMs, and total oxidant status, TOS) and nitrosative (NO⚫ metabolites, NOx) stress, and four methods for the evaluation of the antioxidant status: the total antioxidant capacity (TAC) based on the ferric reducing ability of plasma (FRAP), the total antioxidant activity (TAA) based on the reduction of the coloured ABTS⚫+ radical cation, the free radical scavenging activity (FRSA) based on the reduction of the purple DPPH⚫, and the total thiol levels (TTLs) based on their interaction with DTNB to form a highly coloured anion. Besides, myeloperoxidase (MPO) and ceruloplasmin oxidase (CP) activities were also assessed. Except for TAA, analytical data showed a great inter-individual variation for both oxidant and antioxidant assays. ROMs were strongly correlated with CP, while TOS with MPO and TAC. Furthermore, a tendency between TOS and FRSA was shown. NOx was correlated with TAC and TAA, and a tendency with TOS was shown. No correlations appeared among the antioxidant assays, even if a tendency between TAC and TAA was evidenced, but TAC was correlated with MPO activity. The observed correlation between ROMs and CP is discussed as a possible analytical interference. The absence of correlation among the antioxidant biomarkers suggests the simultaneous use of a panel of tests to verify any changes in the redox balance, mainly in livestock in which reference values for each biomarker are lacking.

Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H2O2. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn2+ and Cu2+. The kinetic parameters Km and Vmax were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the Tm, Tagg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.

A surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) is described for the quantitative analysis of the proteinic stroke biomarker S100-β that has to be detected at very low concentration levels. The Raman reporter 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) on gold nanoparticles (GNPs) was employed as the SERS tags. They are shown to perform much better than bare GNPs in LF strips. The S100-β protein can be detected by this method with very low detection limits by monitoring the intensity of the characteristic Raman peak of the S100-β protein-conjugated GNPs at 1332 cm−1. Under optimized conditions, the assay works in the 1 pg·mL−1 to 40 ng·mL−1 S100-β concentration range, and the detection limit is as low as 0.14 pg·mL−1. This is lower by a factor of 3 compared to colorimetric or fluorimetric methods.

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

Polychlorinated bisphenols (PCBs) continue to contaminate food chains globally where they concentrate in tissues and disrupt the endocrine systems of species throughout the ecosphere. Hydroxylated PCBs (OH-PCBs) are major PCB metabolites and high-affinity inhibitors of human estrogen sulfotransferase (SULT1E1), which sulfonates estrogens and thus prevents them from binding to and activating their receptors. OH-PCB inhibition of SULT1E1 is believed to contribute significantly to PCB-based endocrine disruption. Here, for the first time, the molecular basis of OH-PCB inhibition of SULT1E1 is revealed in a structure of SULT1E1 in complex with OH-PCB1 (4ʹ-OH-2,6-dichlorobiphenol) and its substrates, estradiol (E2), and PAP (3’-phosphoadenosine-5-phosphosulfate). OH-PCB1 prevents catalysis by intercalating between E2 and catalytic residues and establishes a new E2-binding site whose E2 affinity and positioning are greater than and competitive with those of the reactive-binding pocket. Such complexes have not been observed previously and offer a novel template for the design of high-affinity inhibitors. Mutating residues in direct contact with OH-PCB weaken its affinity without compromising the enzyme’s catalytic parameters. These OH-PCB resistant mutants were used in stable transfectant studies to demonstrate that OH-PCBs regulate estrogen receptors in cultured human cell lines by binding the OH-PCB binding pocket of SULT1E1.

Spectroscopy based on surface enhanced Raman scattering (SERS) is widely used as a method with extremely high sensitivity for molecular and chemical analysis. We have developed thin-film sandwich structures, in which, when used as sensitive elements for detecting organic compounds at low concentrations, high-amplitude spectra of surface enhanced Raman scattering are observed. Using gas-phase cryochemical synthesis and thermal sputtering in vacuum, SERS active sandwich structures Ag&ndash, poly(chloro-p-xylylene)&ndash, Ag (Ag&ndash, PCPX&ndash, Ag) were obtained. In the process of creating sandwich structures, the upper silver film takes the form of a complex island topology with submicron sizes. A series of samples were made with different thicknesses of the polymer and upper silver layers. SERS spectra of the analyte chemically adsorbed on the film surface were obtained, demonstrating a significant amplification (up to 104) compared with the control sample. The dependence of the gain on the silver concentration is characterized by a maximum polymer layer thickness of 600 nm and a 30 nm thick upper silver layer. A selective amplification of the low molecular weight compound spectra with respect to proteins was observed. A semi-empirical model is proposed that is in good agreement with the experimental results.

In the present study, surface-enhanced Raman scattering–based lateral flow assay (SERS-LFA) strips were applied to promptly and sensitively detect Escherichia coli O157:H7 (E. coli O157:H7) to ensure food safety. The SERS nanotags were prepared by connecting peculiar monoclonal antibody (McAb) against E. coli O157:H7 directly onto the surfaces of gold-silver core-shell nanostructures loaded with two-layer Raman reporter molecules of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The Raman signal intensity at 1335 cm−1 on the test line (T line) of SERS-LFA strips was detected in the wide range of 101–109 colony-forming units/mL (CFU/mL), and regression models based on machine learning were combined to accurately and quantitatively analyze E. coli O157:H7. The limit of detection (LOD) of the extreme gradient boosting regression (XGBR) based on the Raman signal intensity of DTNB was 6.94 × 101 CFU/mL for E. coli O157:H7, which was approximately four orders of magnitude lower than that of visual limits. In addition, although E. coli O157:H7 was spiked into the food matrices including milk and beef at an ultra-low dose of 10 CFU/mL, the SERS-LFA combined with XGBR was able to successfully explore E. coli O157:H7 from the mixture that was incubated for only 2 h, in which the recoveries were mainly distributed between 86.41 and 128.25%. In summary, these results demonstrated that the SERS-LFA had a significant potential as a powerful tool for the point-of-care testing (POCT) of E. coli O157:H7 in the early food contamination stage.

A series of vinyl sulfone molluscicidal agents was tested to determine the ability to interfere with: a) the color-forming reaction between cysteine and 5,5 1 -dithiobis {2-nitrobenzoic acid) {DTNB); b) the activity of malic dehydrogenase, a typical sulfllydryl-dependent enzyme; c) the in vitro endogenous respiration in the snail, Australorbis glabratus; and d) the in vivo and in vitro endogenous respiration and pyruvate metabolism of liver, brain, and kidney of albino rats. An approximate intraperitoneal LD~o was :J determined for vinyl sulfone in male and female albino rats. The effect of vinyl sulfone on cysteine treated albino rats · was also observed. Vinyl sulfone and derivatives were found to inhibit the color-forming reaction between cysteine and DTNB indicating sulfhydryl reactivity. A positive correlation between this in vitro sulfhydryl reactivity and molluscicidal potency was observed. However, no significant effect on endogenous respiration was observed in A. glabratus . Similarly, no significant effects on endogenous respiration or pyruvate metabolism were observed in the tissues tested from male and female albino rats. High concentrations of vinyl sulfone '1ere required to significantly inhibit the activity o f malic dehydrogenase.

© 2021 Elsevier B.V.In this study, a capillary driven microfluidic chip-based immunoassay was developed for the determination of Human Chorionic Gonadotropin (hCG) protein, which is prohibited by the World Anti-Doping Agency (WADA). Here, we used antibody modified magnetic metal organic framework nanoparticles (MMOFs) as a capture prob in urine sample. MMOF captured hCG was transferred in a capillary driven microfluidic chip consisting of four chambers, and the interaction of MMOF with gold nanorods labelled with 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) as a Raman label was carried out in the capillary driven microfluidic chip. The movement of MMOF through first chamber to the last chamber was achieved with a simple magnet. In the last chamber of capillary driven microfluidic chip, SERS signals of DTNB molecules from the sandwich complex were recorded using a Raman spectrophotometer. The selectivity of the developed method was demonstrated by applying the same procedure for the detection of Human Luteinizing Hormone (hLH), Human Chorionic Gonadotropin Hormone (hGH) and Immunoglobulin G (IgG) protein. The regression coefficient and limit of detection obtained from the standard addition method were found as 0,9985 and 0,61 IU/L, respectively. Furthermore, the conventional ELISA method confirmed that the results obtained by the presented method were acceptable with the similarity of 97.9% in terms of average recovery value, for the detection of hCG in urine samples. The analysis system developed for target proteins will be an alternative technique such as Western Blot used in routine analysis that is expensive and time consuming.

Mercury is one of the more common and potentially most harmful toxic metals. Remediation using conventional physical and chemical methods is uneconomical and generates large volumes of chemical waste. Bioremediation of hazardous metals has received considerable and growing interest over the years. In the present work, genetically engineered Escherichia coli cells, which express four rice metallothionein (MT) isoforms as fusions with glutathione-S-transferase (GST), were tested for their ability to remove mercury. The results showed that the E. coli cells expressing OsMT1, OsMT2, OsMT3, and OsMT4 are able to remove 20, 13.7, 10, and 7 nmol Hg2+/mg (dry weight) from the culture medium, respectively. The recombinant GST–OsMTs were purified using affinity chromatography. The UV absorption spectra and the results of 5,5-dithio-bis-(2-nitrobenzoic) acid (DTNB) assay recorded after the reconstitution of the apo-OsMTs with mercury confirmed that the different OsMT isoforms were able to form mercury complexes in vitro with different binding capacities and different binding strength.

We present an attractive model of surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) for the sensitive and accurate detection of Mycoplasma pneumoniae (MP) infection in human serum. The SERS-LFIA strip uses Au@Ag nanoparticles (Au@Ag NPs) loaded with two layers of Raman dye 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as SERS tags. The advantages of the dual dye-loaded SERS tags (Au/DTNB@Ag/DTNB) are the high sensitivity and the bioconjugation flexibility of the detection antibody. As determined from our SERS-LFIA strip, human IgM was quantified by monitoring the SERS signal on the test line. The limit of detection for human IgM was 0.1 ng mL−1, which was 100 times more sensitive than that by using the colorimetric method. Our assay results for 20 MP-specific IgM positive serum specimens showed 100% accuracy and detection rate, whereas the parallel enzyme-linked immunosorbent assay only showed 85% detection rate. The SERS-LFIA strip also exhibited high specificity and potential clinical applications. Therefore, our SERS-based LFIA strip has strong potential for practical applications in the sensitive and rapid detection of MP.

In this paper, 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) was used as an organic self-assembled monolayer on copper surface for the first time. The inhibition performance in 3.5 wt% NaCl solution was studied via electrochemical techniques, surface analysis methods and theoretical calculations. The influence of concentration on inhibition performance of DTNB was investigated by electrochemical impedance spectroscopy and potentiodynamic polarization. It is found that the inhibition efficiency of the self-assembled monolayer first increases and then decreases with the addition of DTNB, and the maximum inhibition efficiency is 92.4% at the DTNB concentration of 1 × 10-3 mol·L-1. The inhibition effect of DTNB was also evaluated by morphology observation of copper using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Raman spectroscopy, X-ray photoelectron spectroscopy and quantum chemical calculations were utilized to determine that DTNB is well bound to copper surface through chemical bonds.

Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H2O2 treated conditions. Both drtrx1 and drtrx2 genes were induced following H2O2 treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H2O2, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

Background and Objective: Reactive sulphydryl groups in haemoglobin has been related with oxygen binding. In this study, the researchers analyzed the reactive sulphydryl groups in horse (Equus ferus caballus) carbonmonoxyhaemoglobin. Materials and Methods: Hemolysate gotten from fresh horse blood was converted to yield the carbonmonoxy derivative. It was then separated via carboxymethylcellulose into major and minor fractions of haemoglobin. These fractions were titrated with Ellmanʼs reagent (DTNB) and p-hydroxymercuri(II)benzoate (p-MB) stock solutions in increasing volumes. Results: Results showed that two sulphydryl groups reacted with DTNB and p-MB in both major and minor haemoglobin fractions. p-MB is known to be more reactive with thiols than DTNB, on the other hand the reactivity is the same with horse carbonmonoxyhaemoglobin. Conclusion: This study will enable a better understanding as regards the kinetics and equilibrium behind this reaction.

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s-1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils.

SummaryBackground The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. Aim In this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. Methods Whole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. Results We show that the reducing agent dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current–voltage relationship by 23 ± 4.31 mV, suggesting a change in ion selectivity. Conclusion Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg(2+) effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F1FO-ATPase (F-ATPase) activity promoted by micromolar Hg(2+) concentrations were investigated. Hg(2+) was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg(2+) doses which inhibited the NADH-O2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg(2+) dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg(2+) interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria.

Methylmercury (MeHg) is a highly neurotoxic compound to which human populations are exposed via fish consumption. Once in cells, MeHg actively binds thiols and selenols, interfering with the activity of redox enzymes such as thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR) which integrate the thioredoxin system. In fact, it has been shown that inhibition of this system by MeHg is a critical step in the unfolding of cell death. Current clinical approaches to mitigate the toxicity of MeHg rely on the use of chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) which largely replaced British anti-Lewisite or 2,3-dimercapto-1-propanol (BAL) as the prime choice. However, therapeutic efficacy is limited and therefore new therapeutic options are necessary. In this work, we evaluated the efficacy of a macrocyclic chelator, 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S), in preventing MeHg toxicity, namely by looking at the effects over relevant molecular targets, i.e., the thioredoxin system, using both purified enzyme solutions and cell experiments with human neuroblastoma cells (SH-SY5Y). Results showed that [15]aneN4S had a similar efficacy to DMSA and BAL in reversing the inhibition of MeHg over purified TrxR and Trx by looking at both the 5,5&prime, dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay and insulin reduction capability. In experiments with cells, none of the chelating agents could reverse the inhibition of TrxR by MeHg, which corroborates the high affinity of MeHg to the selenol in TrxR active site. [15]aneN4S and BAL, unlike DMSA, could prevent inhibition of Trx, which allows the maintenance of downstream functions, although BAL showed higher toxicity to cells. Overall these findings highlight the potential of using [15]aneN4S in the treatment of MeHg poisoning and encourage further studies, namely in vivo.

In recent years, rapid and accurate detection of pathogenic bacteria has been a current research area in terms of public health and food safety. One of the effective methods for this purpose is Surface-enhanced Raman scattering (SERS), its sensitivity, signal amplification capability, portability, and paper-based horizontal flow immunoassay (LFIA) system; Due to their high analysis rate and low cost, they stand out as a powerful and effective technique for the detection of pathogenic microorganisms. In the first step of this study, E. coli and S. enteritidis strains selected and known common pathogenicity were used to generate the SERS-based LFIA system using the raman tag DTNB; 5,5 ́-Dithiobis(2-Nitrobenzoic acid). Quantitative analysis and rapid detection of selected bacteria was performed after pre-enrichment with Fe3O4/Au-PEI nanoparticles. For this purpose, 20 nm gold nanoparticle (AuNP) and 15 nm Fe3O4/Au-PEI were synthesized. In the next stage, optimized conditions for detection of selected microorganisms were investigated by using rennet enzyme cleaving casein. iv In the second stage of our study, Fe3O4/Au-PEI nanoparticles coated selected bacterial antibody were interacted with E. coli concentrations of 101-107 cfu/mL with used of enzyme based magnetic extraction LFIA system. Then, when the casein was hydrolyzed with the help of rennet, E. coli bound to Fe3O4/Au-PEI nanoparticles was separated by magnetic field. Free bacteria were carried out on nitrocellulose membrane with DTNB raman label bound to AuNPs on conjugation pad (AuNP DTNB antibody complex), and the paper-based LFIA system was then allowed to be detected bacteria concentration on the test line of the nitrocellulose membrane. Tracking the SERS signals of DTNB molecule, LOD: 0,52 cfu/mL and R2: 0,98 values were determined from a linear calibration curve against the increasing concentration of bacteria. These values have been shown to work exclusively for E. coli against different bacterial species such as B. subtilis, M. luteus, S. enteritidis strains. In the third stage of our study, synthetic urine, reference blood and commercial milk samples were used as biological samples. With the developed system, the efficiency of binding of selected E. coli was determined as 86% or more. In the last stage of our study, the effectiveness of E. coli was investigated in synthetic urine, reference blood and commercial milk known as biological samples. Here, this study showed that the synthetic urine, reference blood and milk, valuable nutrients especially indicator E. coli tested as biological samples confirmed the idea that we will detect other pathogen bacteria. In this study, an alternative method was considered due to the expensive antibody used in the determination of pathogen microorganisms. In accordance with this purpose, instead of the antibody used in LFIA system, it is aimed to use bacteriophages as an alternative method because of their natural affinity and high specificity of host selectivity. For this reason, the P22 phage selecting S. enteritidis as the host, and its antibody were used to compare. As a result of bacteriophage study, S. enteritidis (101-107 cfu/mL) was determined from SERS calibration curve as LOD: 7 cfu/mL and R2: 0,98 whereas LOD: 6 cfu/mL and R2: 0,98 were found in our antibody study. These results showed that bacteriophage would be an alternative and inexpensive method compared to antibody in the detection of pathogenic microorganisms. In our in vivo study, egg and chicken samples infected with S. enteritidis were found to bind and separate 90% or more bacteria when using our P22-bacteriophage-based LFIA system. As a result, it was found that our LFIA system works effectively and also bacteriophages can be an alternative method. ABSTRACT ........................................................................................................ iii TEŞEKKÜR ........................................................................................................ vi İÇİNDEKİLER .................................................................................................... vii ŞEKİLLER DİZİNİ ................................................................................................ x ÇİZELGELER DİZİNİ ....................................................................................... xiv SİMGELER VE KISALTMALAR ........................................................................ xv 1. GİRİŞ ........................................................................................................ 1 2. GENEL BİLGİLER .................................................................................... 4 2.1. Bakterilerin Yapısı .................................................................................... 4 2.2. Bakteriyofajlar Giriş .................................................................................. 6 2.2.1. Bakteriyofajların keşfi ........................................................................... 7 2.2.2. Bakteriyofajların yapısı ......................................................................... 9 2.2.3. Bakterilerin tespitinde fajlar ................................................................ 10 2.3. Faj Enfeksiyonu ve Yaşam Döngüsü ...................................................... 11 2.3.1. Litik (parçalayıcı) faj ........................................................................... 11 2.3.2. Ilıman Faj............................................................................................ 12 2.3.3. Faj Enfeksiyon Aşamaları ................................................................... 12 2.4. Salmonella faj ......................................................................................... 14 2.5. Yüzeyde Güçlendirilmiş Raman Spektroskopisi (SERS) ........................ 14 2.6. Patojen bakteri tespiti için SERS etiket yöntemleri ................................. 16 2.6.1. SERS etiketleri ................................................................................... 16 2.6.2. Bakterilerin tespiti için SERS etiketleri ................................................ 17 2.6.3. Bakteri tespiti için etiketsiz yöntem ..................................................... 20 2.6.4. Patojen bakterilerin SERS profili ........................................................ 20 2.6.5. Yeni etiketsiz SERS yöntemleri .......................................................... 21 2.6.6. Nanoyapılı metal yüzeye dayalı SERS yöntemi. ................................ 22 2.6.7. Soy metal NP'lerine dayalı SERS yöntemi. ........................................ 22 2.7. Bakterilerin tespiti için çok fonksiyonlu SERS platformu ......................... 24 viii 3. DENEYSEL ÇALIŞMALAR .................................................................... 27 3.1. Kimyasal Maddeler................................................................................. 27 3.2. Kullanılan Cihazlar ................................................................................. 27 3.3. Kullanılan Diğer Malzemeler .................................................................. 27 3.4. Kullanılan Yöntemler .............................................................................. 28 3.4.1. Çözeltiler ............................................................................................ 28 3.4.2. Benzalkonyum Klorür Kullanılarak Altın Nanopartikül Sentezi ........... 28 3.4.3. Etiket Olarak Kullanılacak Küresel Sitratlı Altın Nanopartikül Sentezi ve Yüzey Modifikasyonu ........................................................................ 30 3.4.4. Sentezlenen Altın Nanopartiküllerin SERS Etkileri ............................ 31 3.4.5. Altın Kaplı Demir Oksit Manyetik Nanopartikül Sentezi Ve Yüzey Modifikasyonu ........................................................................................ 32 3.4.6. Demir Manyetik Nanopartiküllerin Altın ve Polietilen İmin ile Kaplanması: ........................................................................................... 33 3.4.7. Farklı Raman Etiketlerinin AuNP Konjugasyonunda Optimizasyonu . 33 3.4.8. Yatay akışlı immunoassay striplerinin inşası ...................................... 35 3.4.9. Yatay akış immunoassay platformu için test membranı belirlenmesi . 36 3.4.10. Altın nanopartiküllerin agregasyon testi ............................................. 37 3.4.11. Kağıt tabanlı yatay akış immunoassay sisteminin inşa çalışmaları .... 38 3.4.12. Yatay akış immunoassay platformunun E. coli tayininde yapay idrar, referans kan ve ticari süt örneklerinde uygulanması .............................. 40 3.4.13. Manyetik nanoekstraksiyon sonrası E. coli için Yatay Akış İmmunoassay Yöntemi ........................................................................... 41 4. SONUÇLAR VE TARTIŞMA .................................................................. 44 4.1. Manyetik ekstraksiyona dayalı kağıt tabanlı LFIA sistem ....................... 44 4.1.1. Benzalkonyum klorür temelli altın nanopartikül karakterizasyonu ...... 44 4.1.2. Sitrat varlığında AuNP sentezi ve optimizasyon çalışmaları .............. 46 4.1.3. Fe3O4/Au-PEI partiküllerinin sentez ve karakterizasyonu .................. 47 4.1.4. Nitroselelüoz membranda gözenek büyüklük etkileşimleri ................. 52 4.1.5. Altın nanopartikül agregasyon testi .................................................... 53 4.1.6. Farklı Raman Etiketleri ile altın nanopartikül modifikasyonu .............. 55 4.1.7. Manyetik ekstraksiyona dayalı yatay akış immünoassay çalışmaları . 56 ix 4.1.8. Kuantum noktacıkların E. coli antikoru ile modifikasyonu ve enzim ile bölünmesi ............................................................................................... 56 4.1.9. Manyetik nanopartiküllerin E. coli antikoru ile modifikasyonu ve enzim ile bölünmesi ........................................................................................... 60 4.1.10. Manyetik ekstraksiyon ile geliştirilmiş kağıt bazlı LFIA parametrelerinin optimizasyonu ......................................................................................... 61 4.1.11. Manyetik ekstraksiyona dayalı yöntem için kalibrasyon çalışması ..... 65 4.1.12. Geliştirilen kağıt tabanlı LFIA sisteminin örnek denemeleri ................ 68 4.1.13. Manyetik ekstraksiyona dayalı yöntem için seçicilik çalışmları ........... 70 4.1.14. Manyetik ekstraksiyona dayalı yöntem için farklı türdeki manyetik partiküllerin kullanılması ......................................................................... 72 4.2. Bakteriyofaja dayalı kağıt tabanlı LFIA sistem ........................................ 73 4.2.1. Bakteriyofaj bazlı kağıt tabanlı yatay akış immünoassay çalışmaları . 73 4.2.2. İmmünomanyetik ayırım ile S. enteritidis tespiti .................................. 74 4.2.3. Farklı yüzey modifikasyonları ile S. enteritidis tespiti .......................... 75 4.2.4. Faj bazlı ELISA plaka ile bakteri tayini ............................................... 77 4.2.5. Bakteriyofaj bazlı kağıt tabanlı LFIA sistem ........................................ 78 4.2.6. Bakteriyofaj tabanlı LFIA sistemin seçicilik çalışmları ......................... 81 4.2.7. Bakteriyofaj tabanlı sistemin gerçek örnek denemeleri ...................... 83 5. YORUM .................................................................................................. 89 6. KAYNAKLAR ........................................................................................ 100 EKLER ............................................................................................................ 115 6.1. EK 1 – Manyetik partiküllerin FTIR sonucu. .......................................... 115 EK-2 Saf kazein ve manyetik partikül ile kovalent bağlı olan kazein’in FTIR sonuçları ............................................................................................... 116 6.2. EK 2 - Tezden Türetilmiş Yayınlar ........................................................ 117 6.3. EK 3 - Tezden Türetilmiş Bildiriler ........................................................ 118 6.4. EK 6 - Tez Çalışması Orjinallik Raporu ................................................ 119 ÖZGEÇMİŞ ..................................................................................................... 120 Son yıllarda patojen bakterilerin hızlı ve doğru tespiti, halk sağlığı ve gıda güvenliği açısından oldukça güncel bir araştırma alanıdır. Bu amaca yönelik etkin yöntemlerden biriside, Yüzeyde geliştirilmiş Raman saçılması (SERS) olup, duyarlılığı, sinyali çoğaltma yetkinliği, taşınabilirliği ve kâğıt tabanlı yatay akış immünoassay (LFIA) sistem, yüksek analiz hızı, aynı zamanda düşük maliyetli olması nedeniyle patojen mikroorganizma tespitinde güçlü ve etkili bir teknik olarak öne çıkmaktadırlar. Yapılan bu çalışmanın birinci aşamasında, yaygın patojenitesi bilinen ve seçilen E. coli ve S. enteritidis suşları, SERS tabanlı LFIA sistemi oluşturmak için, raman etiketi DTNB; 5,5 ́-Dithiobis(2-Nitrobenzoik asit) kullanıldı, ikinci aşamada da Fe3O4/Au-PEI nanopartikülleri ile ön-zenginleştirme yapıldıktan sonra, seçilen patojen bakterilerin tespiti ve kantitatif analizi gerçekleştirilmiştir. Bu hedefe yönelik olarak, 20 nm boyutunda altın nanopartikül (AuNP) ve 15 nm boyutunda Fe3O4/Au-PEI sentezlenmiştir. Daha sonraki aşamada da kazeini parçalayan rennet enzimi kullanılarak seçilen mikroorganizmaların tespitinde optimize koşullar araştırılmıştır. Araştırmamızın ikinci aşamasında, enzim tabanlı manyetik ekstraksiyon sistemi kullanılarak, seçilen bakteri antikoru ile kaplı Fe3O4/Au-PEI nanopartiküller, 101-107 ii kob/mL E. coli derişimleri olacak şekilde etkileştirilmiştir. Daha sonra rennet yardımıyla kazein hidroliz edildiğinde, Fe3O4/Au-PEI nanopartiküllerine bağlı bulunan E. coli manyetik alan yardımıyla ayrılmıştır. Serbest bakteriler konjugasyon ped (AuNP+DTNB+antikor kompleksi) üzerinde altın nanopartiküllere bağlı olan raman etiket DTNB ile nitroselüloz membran üzerinde yürütülmüş, ve bakteri derişimi kağıt tabanlı LFIA sistem nitroselüloz membran üzerinde bulunan test çizgisi ile tespit edilmesi sağlanmıştır. DTNB molekülünün SERS sinyallerini takip edilerek bakterinin artan konsantrasyonuna karşı lineer bir kalibrasyon eğrisinden LOD: 0,52 ve R2 değerleri de 0,98 olarak saptanmıştır. Bu değerler farklı bakteri türlerine B. subtilis, M. luteus, S. enteritidis suşlarına karşı sadece E.coli‘ye spesifik olarak çalıştığı gösterilmiştir. Çalışmamızın üçüncü aşamasıda, biyolojik örnek olarak sentetik idrar, referans kan ve ticari süt örnekleri kullanılmıştır. Geliştirilen sistem ile seçilen E. coli’yi ne kadar tuttuğumuzun etkinliği %86 ve üzeri olarak saptanmıştır. Yaptığımız bu çalışmada patojen mikroorganizmaların tayininde kullanılan antikorun pahalı olması nedeniyle alternatif bir yöntem düşünülmüştür. Bu amaca uygun olarak, LFIA sistemde kullanılan antikor yerine, doğal afiniteleri ve yüksek özgüllükte konak seçicilikleri nedeniyle bakteriyofajların alternatif yöntem olarak kullanılması hedeflenmiştir. Bunun için S. enteritidis’i konak olarak seçen P22 fajı ve kıyaslamak amacıyla antikoru kullanılmıştır. Yapılan bakteriyofaj çalışması sonucunda S. enteritidis (101-107 kob/mL) SERS kalibrasyon grafiklerinden LOD: 7 kob/mL ve R2: 0,98 olarak saptanırken, antikor çalışmamızda LOD: 6 kob/mL ve R2: 0,98 bulunmuştur. Bu sonuçlar göstermiştir ki patojen mikroorganizmaların tayininde bakteriyofajın antikora kıyasla alternatif ve ucuz bir yöntem olacağı görülmüştür. In vivo çalışmamızda S. enteritidis ile enfekte edilen yumurta ve tavuk eti örnekleri, P22-bakteriyofaj tabanlı yatay akış sistemimiz kullanıldığında, %90 ve üzerinde bakteriyi tuttuğu ve ayırdığı saptanmıştır. Sonuçta yatay akış sistemimizin etkin çalıştığı ayrıca bakteriyofajların alternatif bir yöntem olabileceği görülmüştür.