[Show abstract][Hide abstract] ABSTRACT: Gold nanorods have been nominated as propitious candidates for nanobiodiagnostic applications. Herein, a technique has been introduced for rapid visual detection of lysozyme, as its high level of excretion in biological fluids is a characteristic sign of leukemia and kidney disorders. Gold nanorods were bio-functionalized with lysozyme aptamer and characterized with UV–Visible and FTIR spectroscopy, zeta potential analyzer and transmission electron microscopy. Exposure of the nanoprobe to nano molar levels of lysozyme (20 nmol L À 1) lead to dictated aggregation of the nanostructures at ambient temperature ; which was significantly improved by heat induced morphological perturbations and rapid detection by the naked eye (down to pico molar level). Qualitative analysis of Acute myeloid leukemia, Acute lymphocytic leukemia and Lymphoma blood serums showed sensitivity and specificity of the fabricated aptasensor under both temperature conditions. This report encourages utilization of heat-induced ag-gregation of gold nanorods as a promising nanodiagnostic technique for the emerging nanotechnologies.
[Show abstract][Hide abstract] ABSTRACT: We used a combination of protein engineering and spectroscopic methods to investigate the effect of a long length loop on the conformational stability and activity of chondroitinase ABC I. This study involves manipulation of interactions around Asp(689) as a key residue in the central region of the loop containing residues 681-695 located at C-terminal domain of the enzyme. According to the equilibrium unfolding experiments and considering thermodynamic m value and ΔG(H2O), we found that the folded state of H700N, L701T, and H700N/L701T are more compact relative to the folded state of wild-type protein and they become stabilized upon mutation. However, the compactness and stability of other variants are less than those of wild-type protein. According to enzyme activity measurements, we found that the catalytic efficiency of structurally stabilized variants is decreased, while that of destabilized mutants is improved.
[Show abstract][Hide abstract] ABSTRACT: امروزه ساخت نانوذرات با شکل کنترل شده، توجه دانشمندان بسیاری را در حوزه نانوزیست فناوری به خود اختصاص داده است.
نانومیله طلا با شکل منحصر ب هفرد، خواص نوری ویژهای
دارد که حاصل تشدید پلاسمون سطحی موضعی در بعد طولی نانومیله طلا
است. حساسیت بالای نوسانات پلاسمون طولی این نانوذره نسبت به تغییرات جزیی در محیط پیرامون، آنرا
انتخاب مناسبی در
طراحی ز یحسگرها معرفی نموده است. از سوی دیگر، برای ساخت هر ز یحسگری مقاومت و پایداری اجزای سازنده آن طی فرایند
تشخیص از اهمیت ب هسزایی برخوردار است. این پژوهش، به بررسی پایداری نانومیله های طلا در شرایط دمایی مختلف و احتمال
حفظ مورفولوژی آن م یپردازد.
مطالعات انجام شده نشان داد که بر اثر اعمال حرارت در دماها و زمان های مختلف مورفولوژی میله ای
محفوظ مانده و نانومیلهطلا
از مقاومت حرارتی بالایی برخوردار است. نتایج این پژوهش، بر کاربردپذیری
نانومیله های طلا در طراحی نسل جدیدی از زی حسگرهای مبتنی بر نوسانات پلاسمون سطحی مهر تایید م یزند.
[Show abstract][Hide abstract] ABSTRACT: G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV-Visible spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures.
International Journal of Biological Macromolecules 09/2014; 72. DOI:10.1016/j.ijbiomac.2014.09.016 · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Co-deposition of RF-sputtering and RF-PECVD from acetylene gas and Au target were used to prepare sensor chip of gold nanoparticles (Au NPs). Deposition conditions were optimized to reach a Localized Surface Plasmon Resonance (LSPR) sensor chip of Au NPs with particle size less than 10 nm. The RF power was set at 180 W and the initial gas pressure was set at 0.035 mbar. Transmission Electron Microscopy (TEM) images and Atomic Force Microscopy (AFM) data were used to investigate particles size and surface morphology of LSPR sensor chip. The Au and C content of the LSPR sensor chip of Au NPs was obtained from X-ray photoelectron spectroscopy (XPS). The hydrogenated amorphous carbon (a-C:H) thin film was used as intermediate material to immobilize Au NPs on the SiO2 substrate. The interaction between two types of osmolytes, i.e. sorbitol and trehalose, with Pseudomonas cepacia lipase (PCL) were detected by the prepared LSPR biosensor chip. The detection mechanism is based on LSPR spectroscopy in which the wavelength of absorption peak is sensitive to the refractive index of the environment of the Au NPs. This mechanism eliminates the use of a probe or immobilization of PCL on the Au NPs of LSPR sensor chip. The interaction between PCL and osmolytes can change refractive index of the mixture or solution. We found that unlike to trehalose, sorbitol interacts with the PCL. This interaction increases refractive index of the PCL and sorbitol mixture. Refractive index of PCL in the presence of different concentration of sorbitol was obtained by Mie theory modeling of LSPR peaks. This modeling stated that the present LSPR sensor chip has sensitivity as high as wavelength shift of 175 nm per refractive index. Moreover, the detection of such weakly interaction between bio-molecules cannot be achieved by other analysis.
[Show abstract][Hide abstract] ABSTRACT: Linker mediated self-assembly of gold nanoparticles is emerging as an interesting strategy for construction of hybrid nanoscale systems with enhanced plasmonic circular dichroism (PCD) signals. Herein, gold nanorods were assembled via DNA linker to investigate the possibility of signal enhancement. Assemblies were characterized by UV–visible, fluorescence, dynamic light scattering and circular dichroism (CD) spectroscopy. Hybridization of complementary strands resulted in PCD signal enhancement, which was further monitored by the increase of real time PicoGreen fluorescence intensity. Impressively, such changes in the real time fluorescence and plasmonic CD responses could be used as a new detection method for ultrasensitive sensors.
Journal of Physics D Applied Physics 07/2014; 47(31):315401. DOI:10.1088/0022-3727/47/31/315401 · 2.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously reported an increase in the thermal stability of bacterial laccase from Bacillus sp. HR03 using site-directed mutagenesis. Three-dimensional model of this enzyme showed a negative patch in the connecting loop between domains 1 and 2. In the present study, the stability of laccase in organic solvents was improved by introducing nonpolar (E188 → A, I, L and V) and positively charged (E188 → K and R) residues in this region by site-directed mutagenesis. Irreversible thermoinactivation, C50 value, ∆∆G‡ and kinetic parameters of the wild type and its variants were calculated in the presence and absence of various organic solvents (ethanol, methanol, and 1-propanol). All variants showed higher C50 values when compared to the wild type. Nonpolar amino acid substitutions were found to be the most efficient mutants for their remarkable increase in C50 value and a decrease in thermoinactivation rate (ki) in the presence of mentioned solvents. Data showed that replacing a negative residue with hydrophobic residues on the surface of a protein could enhance thermoresistance as well as solvent stability. The stability of the resulting enzymes was dependent on the length of the alkyl chain. Results demonstrated that solvent tolerance was positively correlated with thermal stability. This article is protected by copyright. All rights reserved
Engineering in Life Sciences 07/2014; 14(4). DOI:10.1002/elsc.201300042 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anisotropic nanoparticles have evinced outstanding optical, catalytic and thermal properties in the recent decade, amongst which, gold nanorods (GNRs) have been nominated as good candidates for biosensing, diagnostic, and imaging technologies. Herein, aptamer functionalized GNRs have been used for calorimetric detection of lysozyme. Nanostructures were covalently conjugated with thiolated sequence of lysozyme aptamer, and the characteristic surface plasmon resonance bands were monitored for stability considerations. Upon introduction of analyte, the nanobiosensor started to develop distinct change of colour shade at various concentrations of biomolecule. The colorimetric detection occurred due to the strong sensitivity of GNRs' LSPR to minute changes of local refractive index; where presence of target biomolecule dictates specific aggregation of the nanostructures. This effort encourages fabrication of novel nanorod based aptasensors for the emerging lab-in-a-vial techniques.
TechConnect World 2014, United States of America; 06/2014
[Show abstract][Hide abstract] ABSTRACT: Plasmonic circular dichroism (CD) responses of hybrid nanostructures containing noble metal nanoparticles and chiral molecules have received increasing interest with various applications in nanophotonics. Chiral biomolecules show strong CD signals typically found in the ultraviolet region, whereas, in the visible range, they produce a weak signal. Strengthening the CD signal in the visible region is of high importance, which could be achieved through fabrication of novel hybrid nanostructures. Herein, gold nanoparticles (GNPs) have been assembled via DNA linker to investigate the possibility of enhancing plasmonic CD signal in the visible range. DNA-linked assemblies with pre- and postannealed conditions were characterized by ultraviolet–visible spectroscopy, dynamic light scattering (DLS), and CD spectropolarimetry. In the presence of DNA linker with sticky ends, the aggregation phenomenon was traced by red shifts of surface plasmon resonance of nanoparticles. Time-dependent hybridization of single-stranded “sticky ends” with DNA-conjugated GNPs and increased probability of hydrogen bond formation lead to enhancement of CD signals in the ultraviolet region. Complexation of biomolecule and nanoparticle assemblies induced enhanced CD signals in the visible range, which was noticed both before and after purification. DLS characterization of the assemblies also confirmed the difference in the size of aggregates, which could be controlled by the linker molecules. This investigation encourages possibility of utilizing plasmonic CD technique as a tool for tracing fabricated nanostructure assemblies with enhanced characterization possibility.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Remarkable optical properties of gold nanorods (GNR) have been vastly used in biosensing and biomedical applications. In the light of sensing applicability, GNR based nanobiosensors are functionalized by various biomolecules such as peptides and oligonucleotides, that are known as nanoprobes. However, great sensitivity of GNRs to trace environmental changes and tendency to non-specific aggregation makes a crucial step in design and production of nanoprobe. Therefore, optimization of biorecognition element and stability investigations of nanoprobe is of great significance. Herein, stability of aged samples of oligonucleotide-functionalized GNRs has been studied at ambient temperature.
Method: Gold nanorods were synthesized according to previously reported seed-mediated growth method. The nanostructure was purified by centrifugation and redispersed with a fixed concentration (1 OD). Surface of GNRs was biofunctionalized by a thiolated 22-mer oligonucleotide (1nM) after concentration optimization. Surface plasmon resonance (SPR) and Fourier transform infrared spectroscopy were used to monitor the bioconjugation via formation of Au-S bond. Samples of nanoprobe were then aged at ambient temperature (25 ºC) for different time intervals. Stability of the aged nanoprobe was investigated by monitoring the SPR bands of the nanostructure.
Results: The characteristic SPR bands of GNRs along the short and long axis appeared at 520 nm and 724 nm, respectively. There was negligible shift in the wavelength maxima of nanoprobe after one week aging, with little decrease of intensity for both transverse and longitudinal surface plasmon resonance.
Conclusions: The oligonucleotide-functionalized nanoprobe is stable at ambient temperature for biorecognition purposes.
Keywords Gold Nanorod, Aging Process, Nanoprobe, SPR
1st Tabriz International Life Science Conference and 12th Iran Biophysical Chemistry Conference; 05/2013
[Show abstract][Hide abstract] ABSTRACT: The Cu 2+ dependent DNA cleavage DNAzyme is the unique example of known DNAzymes. The most established method for measuring the kinetics of deoxyribozyme reactions is based on the use of radiolabeled substrates. This method entails the cumbersome sampling, electrophoretic separation, and quantitation of the reaction products, as well as the continuous loss of substrate due to decay of the radioactive label. In this study we represent accurate, fast and inexpensive methods for kinetic study of the DNAzyme which is based on spectroscopic techniques. One of these methods is based on the SYBR gold extrinsic fluorescence. This probe has high affinity to double stranded DNA compared to single stranded DNA. Continuous Hyperchromicity assay (CHA) is used in second method which is a function of DNA hypercromicity that observed when double stranded DNA transformed to single stranded DNA. Using this methods can be traced DNAzyme substrate hybridization and product formation Simultaneously with reaction progress. Under conditions where the product release faster than enzymatic reaction, production release speed which is traced with these methods determined enzymatic reaction kinetic. These methods in addition to the kinetic study of the restriction DNAzyme can be used to study of other DNAzymes that has nucleic acid substrate. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method using SYBR gold because CHA method is DNA intrinsic physicochemical property result without interference of disturbing factors such as SYBR gold.
1st Tabriz International Life Science Conference & 12th Iran Biophysical Chemistry Conference; 05/2013
[Show abstract][Hide abstract] ABSTRACT: Structure-function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp.HR03 with the ability of bilirubin oxidation beside its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp(500) located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase kcat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveals the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged.
International journal of biological macromolecules 05/2013; 60. DOI:10.1016/j.ijbiomac.2013.05.011 · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: در دهه هاي اخير، نانومواد گوناگوني پا به عرصه پژوهش در زمينه هاي پزشکي گزارده اند. نانوميله هاي طلا به دليل دارا بودن خواص اپتيکي منحصر به فرد، گزينه مناسبي جهت امر تشخيص شناخته شده و لذا در ساخت نانوبيوسنسور مورد توجه قرار مي گيرند. در اين راستا، پژوهش حاضر به بررسي پايداري نانوکاوشگر ميله اي طلا پس از اتصال به توالي اليگو نوکلئوتيدي در دماي محيط و دماي بالا پرداخته است. نتايج مطالعات نشان داد که نوسانات پلاسمونيک نانوپروب ميله اي در هر دو شرايط دمايي و در گستره غلظتي پيکومولار از اليگو نوکلئوتيد پايدار مانده و نانوساختارها متجمع نشده اند. دستاورد اين پژوهش، بر پايداري کانژوگه نانوساختار-اليگو نوکلئوتيد براي طراحي نانوبيوسنسورها مهر تأييد مي زند.
اولین همایش ملی و کارگاه های تخصصی علوم و فناوری نانو; 05/2013
[Show abstract][Hide abstract] ABSTRACT: Laccases (benzenediol oxygen oxidoreductases, EC 22.214.171.124) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8M urea and 4mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100mM) containing 4mM ZnSO4 and 100mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.
[Show abstract][Hide abstract] ABSTRACT: The effect of proline on refolding and unfolding kinetics as well as activity of Lipase from Pseudomonas fluorescens was determined using stopped-flow fluorescence and UV/Vis absorbance spectroscopy. Enzyme assay at different concentrations of proline revealed that activity of enzyme reaches maximum at 0.6M concentration of proline. Kinetic measurements showed that refolding rate is considerably accelerated in the presence of 0.6M proline. Unfolding kinetic traces were fitted to double exponential function, and it was revealed that lipase molecules were unfolded via two different pathways. Fast unfolding rate constant decreased, while slow one did not change significantly upon addition of proline.
International journal of biological macromolecules 01/2013; DOI:10.1016/j.ijbiomac.2012.12.046 · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Like many other bacterial lipases Pseudomonas aeruginosa lipase has a very strong structure-function relationship. Herein, the effect of structural compactness of lipase has been investigated on its activity. Pseudomonas aeruginosa HR59 was isolated from burn infection as a new and inexpensive source. Bacterial lipase was extracted from this strain and purified by ion-exchange chromatography. Two divalent metal ions (calcium and magnesium) and three polar organic solvents (methanol, ethanol and iso- propanol) were used to study the structure-function relationship. Results of this study revealed that these additives activated enzyme by reducing the helix content of enzyme. Moreover, thermo stability of the enzyme decreased in the presence of calcium and magnesium ions; whereas it increased upon interaction with polar organic solvents under heat-induced denaturation. Results of this investigation encourage utilization of these activity enhancers for various medical and industrial applications.
International journal of biological macromolecules 12/2012; 54. DOI:10.1016/j.ijbiomac.2012.12.020 · 3.10 Impact Factor