hedayatollah ghourchian

L-Asparaginase converts L-asparagine to L-aspartic acid and causes cancer cells to starve. The main idea of the current study was to improve the biochemical properties of this enzyme using immobilization onto modified magnetic nano-particles (NPs). To this end, Fe3O4 NPs were synthesized, coated with an Au shell, and conjugated with cysteine. The formation of NPs and core-shell structures and their morphology were confirmed using Fourier Transform Infrared spectroscopy (FTIR), Energy Dispersive X-Ray (EDX), VU-Vis, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Also, Circular Dichroism (CD) and fluorescence spectroscopy were employed for the analysis of the secondary and tertiary structures of the immobilized L-ASNase. The alterations in the kinetic parameters of the immobilized enzyme were analyzed using a Lineweaver-Burk plot. The results of instrumental chemistry analysis confirmed the formation of NPs and core-shell structure, and cysteine binding with the core-shell. Based on CD and fluorescence results, no significant changes were observed in the secondary and tertiary structures of the immobilized enzyme compared to the free one. Kinetic parameters of the immobilized enzyme improved compared to the free enzyme so that Km decreased from 4.43±0.05 to 3.75±0.12 mM and Vmax increased from 187.23±11 to 224.78±16 μM min-1mg-1. Also, the stability of the immobilized enzyme improved with acidic and alkaline pH values compared to the free one at temperatures higher than 50 0C. In addition, the reusability of the immobilized enzyme was superior to the free enzyme, with the immobilized enzyme maintaining 72% of its activity after 15 cycles of catalytic reaction. The immobilized enzyme showed an 86% residual activity after 120 min incubation with trypsin, which was higher than the free enzyme (37%). According to the results of this study, immobilization of L-ASNase onto magnetic NPs can be an efficient strategy to enhance the biochemical properties of this enzyme.

Early pregnancy diagnosis is a key factor in shortening the calving interval through identifying the non-pregnant animals to rebreed them at the earliest time after artificial insemination (AI). Bovine pregnancy-associated glycoproteins (PAGs) are produced by mono- and binucleated trophoblastic cells of the pregnant cow’s placenta. Detection of PAG in the maternal circulation has been used to accurately diagnose pregnancy. Several studies have used commercial PAG tests to determine pregnancy status in dairy cows and heifers. Pregnancy-specific protein B (PSPB) or PAG1 was the first identified member of the PAG family and commercial diagnostic kits still utilize PAG1 as a pregnancy marker. In recent years, the use of nanoparticles in immunosensors has been increased their sensitivity and increased the traceability of antigen-antibody responses. Due to the limitation of using ultrasound and other methods for pregnancy diagnosis in the first 30 days after inoculation, the aim of this study was to apply the pregnancy-associated glycoprotein detection method in order to reduce the time of pregnancy diagnosis. For this purpose, a sandwich ELISA immunosensor was designed for the detection of PAGs and named Nano-kit. Magnetic nanoparticles were used to enhance the contact area between antibodies and antigens. Streptavidin and biotin were used for their high binding affinity to bind the antibodies and enzymes to nanomagnet. The synchronization technique and artificial insemination (AI) was performed in Holstein dairy cows (n = 58). Pregnancy status was determined by transrectal ultrasonography (30 days after AI). Furthermore, transrectal palpation was carried out by a skilled veterinarian on day 60 after AI to determine the pregnancy status of cows which were previously detected as pregnant. For PAG1 analysis, blood samples (10 mL) were collected daily from 18 to 30 days after AI. Blood samples were collected by venipuncture from the tail vein into evacuated tubes containing EDTA as an anticoagulant and were processed 1–3 h after sampling. The samples were centrifuged and plasma samples were transferred to fresh tubes and were stored at −20°C until they were assessed. Plasma concentrations of PAG1 were determined by nano- kit and commercial kit. Differences in the level of PAG-1 were evaluated by repeated measures ANOVA (SAS 9.4) over days of sampling (18 to 30). Values of PAG-1 in pregnant cows were considered as the reference. Data were presented as means ± SD and differences were considered significant at P<0.05. By doing Ovsynch protocol, of the 54 cows enrolled in the first AI, 48% (26/54) of synchronized cows were diagnosed pregnant 30 days after AI using transrectal ultrasonography. From day 30 to 60 after AI, 73% (19/26) of cows maintained pregnant and the pregnancy loss from day 32 to 60 after AI was 17% (7/26). Measurement of different concentrations of PAG1 (standards) using a commercial kit and nano-kit showed that nano-kit were more sensitive than the commercial kit and detected a concentration of 0.03 ng/mL. The first increase in plasma concentration of PAG1 occurred on d 23 after AI in pregnant cows and PAG1 concentration in serum increased from d 22 to 30 after AI and it was affected by day (P < 0.001). Cows diagnosed as pregnant on day 60 after AI had a higher PAG concentration on day 24 compared with cows that were diagnosed as non-pregnant (2.28 ± 0.15 ng/mL vs 0.7 ± 0.18 ng/mL, respectively; P < 0.001). Accuracy in predicting pregnancy at day 24 of gestation based on circulating concentration of PAGs was 95 % for 1.93 ng/mL. Generally, based on the results of this study, the cows with mean PAG1 concentration more than 2.28 ± 0.07 ng/mL on 24 and mean PAG1 concentration more than 9.2 ± 0.07 ng/mL on day 30 of pregnancy, remained pregnant until day 60 of pregnancy. Furthermore, in cows diagnosed pregnant on day 24 of pregnancy but were not pregnant on day 30 after AI, blood plasma PAG1 concentration on day 24 after AI was 1.03 ± 0.66 ng/ml. Based on these results, PAG concentrations at day 24 of gestation are higher in pregnant compared to non-pregnant dairy cows and could be applied in diagnosing pregnancy at day 24 of gestation; however, further study is needed to determine the potential of PAG1 in pregnancy diagnosis in dairy cows.
Abortion, Glycoprotein, Nano-kit, Pregnant cow

A biofuel cell is a device for converting chemical energy to electrical energy by a simple way. A high-impact anode is prepared in this research. Here, carboxylated multiwall carbon nanotube (COOH-MWCNT), polydiallyldimethyl ammonium chloride (PDDA) and alcohol dehydrogenase were cast on modified glassy carbon with polymethylene green to construct the bioanode for biofuel cell. The polymethylene green is used as an electron mediator for NAD+ that is coenzyme for alcohol dehydrogenase. This new bioanode construction has a good storage and operational stability. The modified electrode was characterized by cyclic voltammetry. The biofuel cell was made with bioanode and carbon-platinum cathode, with and without membrane, and characterized by linear sweep voltammetry to obtain polarization curve for biofuel cell assembly. The biofuel cells had power density 350 µW.Cm-2 and 1713μW.Cm-2, with membrane and membraneless, respectively. The open circuit voltage of both biofuel cells was 0.28V for more than 1 hour. The anode easy construction and simple cell design make it useful for implant medical instruments.

The Bio-Analysis Lab (with former name of Microanalysis Lab) was established in 1975 at the Institute of Biochemistry and Biophysics, University of Tehran. Many research works and studies have been carrying out in this lab such as electrochemistry of proteins, design and development of nanoparticles for development of novel biosensors, structural analysis of proteins by electrochemistry development of enzyme/microbial biofuel cells. For these purposes, different electrochemical and spectroscopic techniques and various imaging methods were used. In the present report, at first the direct electrochemistry of enzymes such as glucose oxidase, horseradish peroxidase, catalase, euphorbia latex amine oxidase, superoxide dismutase and horse heart cytochrome c were considered. Then, the methods for applying different types of macromolecules such as enzymes, antibodies, nucleic acids and aptamers for developing different types of optical and electrochemical biosensors were illustrated. At the end, some biocompatible nanoparticles for biomedical applications including cancer therapy and also application of some nanomaterials for improving the efficiency of biofuel cells were reviewed.

A feasible and fast method for Adult Hemoglobin (A-Hb) and Fetal Hemoglobin (F-Hb) study was developed by immobilization of Hb on gold-coated magnetic iron oxide nanoparticles (GMNPs).The prepared GMNPs composite nanoparticles with 60 nm diameter were used as a carrier for the immobilization of Hb. The A-Hb and F-Hb were physically attached to the GMNPs nanoparticles. The direct electroche- mistry of F-Hb and A-Hb showed a quasi-reversible cyclic voltammogram corres- ponding to the Heme group with a formal potential of 314 and -334 mV in 0.1M PBS (pH 6.2), respectively. The apparent charge transfer rate constant (ks) and transfer coefficient (α) for electron transfer between the electrode surface and pro- tein were calculated as 0.29/s and 0.1 for F-Hb and 0.21/s and 0.47 for A-Hb. The linear concentration rangesare17.3–225 and 7.4-53 mM for F-H band A-Hb biosen- sors for H O detection. The lifetime of biosensor is more than 2 weeks.

Enzymatic biofuel cells have many great usages as a small power source for medical and environmental applications. In this paper, we employed carboxylated multiwall carbon nanotube- (1-ethyl-3-methylimidazolium bis (trifluoromethyl sulfonyl) imide) ionic liquid nanocomposite on two different electrodes (glassy carbon and carbon felt) for immobilizing alcohol dehydrogenase. The properties of the two types of electrodes were characterized by cyclic voltammetry analysis. Polarization analysis and field emission scanning electron microscopy were used to show differences in the nanobiocomposite immobilization on two electrodes. Compared to glassy carbon, carbon felt achieved much more gains in electrochemical activity and power by catalyst coating. Power density of 10.027μWcm−2, has been achieved by carbon felt, but glassy carbon showed 1.7 μWcm−2 respectively.

Choline oxidase (ChOx) was chosen as a model enzyme for evaluating the performance of CNTs’ functional groups for development of enzyme electrodes. CNTs were functionalized with carboxylic acid, amine or amide groups. Carboxylic acid, amine and amide functionalized CNTs were obtained by acid treatment, ethylenediamine or tetraethylenepentamine chemically modification and ammonia plasma treatment, respectively. The CNTs with different functional groups were mixed with 1-butyl-3-methylimidazolium tetrafluoroborate as a typical room temperature ionic liquid. ChOx was adsorbed on the thus prepared nanocomposites containing different modified electrodes and its electron transfer and electroanalytical response towards choline was investigated. The resulting data showed that the ammonia plasma treated nanocomposite had higher apparent heterogeneous electron transfer rate constant (2.74 s-1) than the others, indicating more facile and rapid rates of electron transfer; while, nanocomposites modified with tetraethylenepentamine functionalized CNTs showed the most sensitive response towards choline (1.09×103 A M-1 m-2) with the lowest detection limit of 5.81×10-6 M. Consequently, tetraethylenepentamine modified electrodes were more convenient for choline biosensing applications.

The ingested nitrates sourced from tap water, food, chemicals and pharmaceuticals are converted to nitrites in the body surfaces by bacteria and then, the nitrite ions can lead the structural changing in hemoglobin. In the present work, aggregation of the purified hemoglobin in adult (HbA) and in fetus or newborn (HbF) in the presence of nitrite ions were studied. Hemoglobin aggregation was performed chemically in the presence of 10 mg/l nitrite ions and examined by UV-Vis spectrophotometer at 360 nm wavelength. The extrinsic fluorimetric measurements indicated that repulsive electrostatic interaction between nitrite anions and negative charged groups of both types of HbA and HbF molecules leads to expose the hydrophobic patch of the protein molecules. Moreover, the α-helix to β-strand transition in both types of hemoglobins shown by circular dichroism support aggregation process among this protein. However, at natural pH, the protonated amino group of Gly in HbF tends to bind to nitrite anions more than the unprotonated forms of Val residue in HbA. The drastic slop of aggregation plot and shorter lag time of HbF relative to HbA demonstrated more aggregation of former protein.

A new signal amplification strategy based on simultaneous application of gold nanoparticles (AuNPs) and horseradish peroxidase (HRP) was employed to improve the sensitivity of an electrochemical immunoassay for detection of human IgG (hIgG), as a model antigenic protein. This immunoassay system was fabricated on magnetic carboxyl-functionalized multi-walled carbon nanotubes (COOH-MWCNT/Fe3O4 nanocomposite). The COOH-MWCNT/Fe3O4 nanocomposite was constructed by chemical co-precipitation of Fe2+ and Fe3+ in alkaline solution in the presence of the COOH-MWCNT. Goat anti-human IgG (anti-hIgG) was covalently bound to the carbon nanotubes and the resulting anti-hIgG bearing nanocomposite was immobilized on the surface of a gold electrode using a permanent magnet. Human IgG (hIgG) as an antigen was detected electrochemically using a secondary HRP-conjugated anti-hIgG immobilized noncovalently on the surface of AuNPs. Electrochemical detection of hIgG was performed in the presence of H2O2 and KI as substrates of HRP. When the concentration of antigen was 200 ng/ml, the sandwich arrangement with AuNPs amplified electrochemical response 56 μA more than a same sandwich arrangement without AuNPs. Binding of HRP-conjugated anti-hIgG to AuNPs was studied by UV–Visible and fluorescence spectrophotometry. The spectrophotometric data showed that binding of antibody molecules to AuNPs occurred without any significant structural changes.

The oriented immobilization of antibody is the most critical step in preparation of a highly sensitive immunosensor. In the present report multiwalled carbon nanotubes were treated with nitric acid followed by ethylenediamine to produce amine functionalized carbon nanotubes. To obtain a properly oriented immobilization, initially the sugar chain of antibody (anti-IgG) was oxidized to aldehyde using periodate and then the aldehyde groups were allowed to react with amine groups of carbon nanotubes. The antibody immobilization was verified using the techniques such as field emission scanning electron microscope, Fourier transform infrared, UV-Vis spectroscopy and electrochemistry. Finally determination of antigen (human IgG) was performed in the presence of conjugated horseradish peroxidase as a label, and H2O2 and potassium iodide as the enzyme substrates. At the optimum pH of 7.2, the detection limit of 16.6 ng/ml was obtained for determination of human IgG.

Cysteine self assembled monolayer-modified gold (Cys/Au) electrode is used to immobilize superoxide dismutase (SOD) and establish a direct electron transfer between enzyme and electrode surface. However, due to the redox activity of copper ion on Cys monolayer, there would be an ambiguity in electrochemical studies of immobilized SOD on Cys/Au electrode. We designed a series of experiments to clarify the role of Cys in this process. Comparison between voltammograms of different electrodes revealed that the current intensity was increasing by the order of Cu+2/Cys/Au>SOD/Cys/Au>Cys/Au, while their electrochemical working windows were overlapping. Furthermore, for these electrodes the electron transfer rate constant were 0.77, 0.73, and 0.29 s-1 and the surface concentration of electroactive species were 1.05×10-10, 1.51×10-11, 1.50×10-11 mol cm-2, respectively. When phosphate buffer solution (PBS) was prepared by ultrapure phosphate salts (copper ion free) no redox response was observed while, by deliberately addition of Cu2+ the Cys/Au electrode showed a redox response. EDTA as chelating agent could pick up Cu2+ from PBS and consequently no electrochemical response was observed for Cys/Au electrode. Comparing these results indicated that the source of the inherent electrochemical activity of Cys/Au electrode is Cu+2. Finally, the Cys/Au electrode was also examined as a sensing system for determination of O2 •−.

Cytochrome c (Cyt c) was absorbed on gold electrode modified with a mixture (1:3) of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid (C11) solutions. The Cyt c/C11/Au electrode thus prepared showed a well-defined quasi reversible voltammetric peak with electron transfer rate constant of 234 s-1. The surface coverage of Cyt c oriented on the surface of C11-modified electrode was determined to be 4.73×10-11 mol cm-2. The Cyt c/C11/Au electrode linearly responded to H2O2 in the concentration range from 90 to 500 μM with a correlation coefficient of 0.9973. Using the reciprocal plot of the steady state current versus hydrogen peroxide concentration, apparent Michaelis constant for the Cyt c was estimated to be 759 μM. In addition, voltammetric analysis was used to study the in situ denaturation of the immobilized Cyt c by gradual addition of either urea up to 8 M or dimethyl formamide (DMF) up to 55%. It was revealed that the mechanism of Cyt c denaturation depends on media (organic or aqueous) were used. So that in the presence of DMF it follows a two-phase process, whereas in urea containing buffer solution, it pursues a single-phase course.

A thin layer of poly methylen green (PMG) was covered on glassy carbon (GC) electrode surface by electrochemical polymerization method. In the next step by dropping a suspension of carboxylic acid functionalized carbon nano-tubes on the PMG/GC electrode a layer of CNTs was coated on the electrode. Thereafter, to immobilize the enzyme on electrode surface, three layers of PMG, alcohol dehydrogenase and PMG were added to the modified electrode, respectively. The Fourier transform infrared, scanning electron microscopy and cyclic voltammetry measurements clearly confirmed the successful immobilization of enzyme on the GC electrode.

Urease was immobilized on platinum electrode both by chemical binding and electropolymerization.The conductometric urea biosensor thus prepared showed a detection limit of 4.9×10-5 M and linear dynamic range from 4.9×10-5 to 5.8×10-3 M for urea concentration when the enzyme is covalently immobilized on Pt electrodes. Conductometric transducers respond to the changes in ionic strength thereby leading to uncontrolled inaccuracies. Such interferences were effectively suppressed by here the use of differential sensor pairs. It was shown that measurements in diluted fluids are possible with the use of a reference sensor having no immobilized enzyme. Specifically, the sensor constructed by covalent binding provided more reproducible responses than that prepared by electropolymerization. Namely the former preserved 80% of its initial activity over a period of 25 days. In order to provide more reproducibility, a method was developed for regeneration of the sensor surface so that, the renewed sensor would responded to urea almost as new.