Tris Buffer pH Research Articles

Fabrication of an IgG-imprinted monolith micro-solid phase extraction chip for IgG extraction from human plasma

Molecular imprinted polymers (MIPs), also known as synthetic antibodies, offer a novel and promising approach for protein separation at analytical scale. In proteomics research, this approach could capture target high abundant proteins from biological fluids to reduce sample complexity. In this study, we have explored the potential to imprint human immunoglobulin G (IgG) to develop a micro-solid phase extraction chip for selective removal of IgG from human plasma. Imprinting of IgG within the polyhydroxy ethyl methacrylate monolith containing dimethyl amino ethyl methacrylate (DMAEMA) as a functional monomer was successfully achieved and characterized using FT-IR spectroscopy and protein assay kit. 0.1 M tris buffer (pH 7) and UV-polymerization were identified as the ideal buffer and polymerization strategy to imprint IgG within 30 min. Template (IgG) elution using 10 % acetic acid containing 10 % (v/v) SDS as elution buffer was moderately successful (25 % IgG-template free monolith was obtained). Protein shape has influenced its imprinting and elution from the monolith. The protocol developed to imprint one protein cannot be translated to another protein without further modifications. A 10-fold decrease of DMAEMA concentration within the monolith and a sequential elution step has resulted in >90 % IgG-template free monolith. The IgG-template free monolith has displayed an adsorption capacity of ∼ 3.7 mg per g monolith and showed good IgG selectivity as compared to other tested human proteins (human serum albumin, transferrin and fibrinogen). Translation of imprinted monolith protocol to develop a micro-solid phase extraction chip was successful. The chip can be re-used for 5 cycles with >95 % reusability and has selectively captured IgG from human plasma.

Standardized Sperm Cryofreezing Technique for Caspian Sea Sturgeons (Huso huso, Acipenser persicus, Acipenser stellatus, Acipenser nudiventris)

Sturgeons, considered `living fossils,` face extinction due to various factors, according to the International Union for Conservation of Nature's Red List. The present study describes a uniform sperm cryopreservation method for the endangered Caspian Sea sturgeon species, which are beluga (Huso huso), Persian (Acipenser persicus), stellate (Acipenser stellatus), and ship (Acipenser nudiventris) sturgeons for both artificial reproduction and ex-situ conservation attempts. For this aim, sperm samples were diluted 1:1 in extender composed of 118 mM Tris buffer (pH=8) and 23.4 mM sucrose-based extender containing 15% DMSO and equilibrated for one hour at 4°C. Then, sperm samples were placed into 0.50-ml straws and multi-stage cooling was applied to freeze with the help of a programmable freezer with an accuracy of ±0.1C/min, and finally stored in liquid nitrogen (-196C) tank. Thawing was performed at 40°C water baths for 25 s. The results indicated that storage for two years resulted in a 24.7% fertilization rate for the stellate sturgeon, while the Persian sturgeon had a remarkable rate of 58.2% after five years storage.

A solid-state sensor based on nanosilica/methylcellulose composite and chromoionophore 8-carboxamidoquinoline derivative as an optode pellet for Zinc(II) ion

Zinc is known to play a central role in the immune system that creates a resistance barrier against viral infection. A naked-eye zinc(II) (Zn2+) ion sensor of 2-oxo-2-(quinolin-8-ylamino)acetic acid (OQAA) as a recognition probe was designed and synthesized. By inserting functional groups of amide and carboxylic acid into a conjugated molecule of 8-aminoquinoline, Zn2+ ion (borderline acid) coordinated favorably with aromatic nitrogen atoms (borderline bases) and N-amide. Characterization of OQAA was done by FTIR, 1H NMR, 13C NMR, UV–vis, and ESI-MS. The Zn2+ ion chemosensor was fabricated by immobilizing OQAA chromogenic ionophore onto a nanosilica/methylcellulose (Si/MC) composite pellet to form a solid-state pellet sensor. A distinguishable color change from pale light-yellow to yellowish-brown of the optode pellet was observed in the presence of Zn2+ ions. Optimization of the pellet-based Zn2+ ion sensor was carried out with a fiber optic reflectance spectrophotometer at 0.25 M OQAA in tris buffer (pH 7.4). The optical fiber sensor demonstrated a linear reflectance response between 0.8 M and 2.4 M Zn2+ ion (R²=0.9741) at λmax=635 nm with a limit of detection (LOD) of 3.6 × 10−2 M and response time of 5 min. The reflectance sensor exhibited good selectivity towards Zn2+ ion over other competitive heavy metals e.g., iron(II) (Fe2+), nickel(II) (Ni2+), cobalt(II) (Co2+), copper(II)(Cu2+) and cadmium(Cd2+) ions. Additionally, the proposed Zn2+ ion sensor showed high shelf-life stability of 75 days operational duration with 80.0 % initial optical response remaining. The quick, easy, and uncomplicated preparation of the OQAA-modified Si/MC (OQAA-Si/MC) composite pellet novel material gave a reproducible reflectance signal towards the determination of Zn2+ ion concentration with a low relative standard deviation (RSD) obtained at ∼1.0 % (n=12), and was reusable three times for assay of Zn2+ ion (RSD=1.3 %). The optical chemosensor imparts remarkable color change towards the analysis of Zn2+ ions, and could offer direct and fast in-situ visual inspection of Zn2+ ion levels in complex samples without an instrument.

Dissolution of Bioactive Glass S53P4 in Continuous Flows of Tris Buffer and Lactic Acid

In vitro dynamic dissolution of bioactive glass S53P4 particles was studied in a cascade of three reactors. Tris buffer (pH 7.40) and lactic acid (pH 2.00) with flow rates of 0.2 and 0.04 ml/min were fed through the reactors for 24 h. The increased ion concentrations in Tris inflows to the second and third reactors decreased the dissolution of the particles. However, the normalised surface-specific mass loss rate decreased from the first to the third reactor and with decreasing flow rate. No distinct differences were observed in the reaction layers on the particles in the three consecutive reactors. This implied that the ions released in the previous reactors contributed to the reaction layers formed in the following reactors. Highly incongruent dissolution with similar dissolution rates of sodium, calcium, and phosphorus occurred with the two flow rates in lactic acid. Although a thick silica-rich layer formed on the particles, the low pH prevented calcium phosphate layer precipitation. The results imply that S53P4 particles in an implant react at different rates depending on their location but form similar reaction layer morphologies independent of their location in physiological solutions (pH 7.4). On the other hand, S53P4 particles exposed to acidic solutions with a pH < 5 likely dissolve incongruently, leaving a slowly dissolving Si-rich layer. In such an environment, the dissolution rates of Na, Ca, and P are independent of the location of the S53P4 particle in the implant. Thus, the pH and fluid flow are critical factors for the dissolution of S53P4 bioactive glass particles.

Triplet state spectroscopy reveals involvement of the buried tryptophan residue 310 in Glyceraldehyde-3-phosphate dehydrogenase (GAPD) in the interaction with acrylamide

Using conventional steady state and time resolved fluorescence study of the interaction between a multi-tryptophan protein and a quencher, it is difficult, if not impossible to identify the particular tryptophan residue/residues involved in the interaction. In this work we have exemplified the above contention using a multi-tryptophan protein, Glyceraldehyde-3-phosphate dehydrogenase (GAPD) from rabbit muscle having three tryptophan (Trp) residues at positions 84, 193 and 310 and a neutral quencher acrylamide in Tris buffer of pH 7.5. From the steady state and time resolved fluorescence quenching (at 298 K) with acrylamide Ksv, K and kq for the system have been calculated. Low temperature phosphorescence (LTP) spectra at 77 K of GAPD in suitable cryosolvent is known to exhibit two (0,0) bands corresponding to two tryptophan residues 193 and 310. Using the LTP study of free GAPD and GAPD – acrylamide it is possible to identify that the buried Trp 310 residue is specifically involved in the interaction with acrylamide. This is possible without doing any site-directed mutagenesis of GAPD which contains Trp residues at 84, 193 and 310. Tyrosine 320 is also specifically quenched. The results have been corroborated using the molecular docking studies. Molecular Dynamics simulation supports our contention of the involvement of Trp 310 and also shows that the other nearest residues of acrylamide are Val175 and Val232.

Synthesis of Ni2+-functionalized polydopamine magnetic beads for facilitated purification of histidine-tagged proteins

Facilitated purification of proteins, at a low cost and a short time, is one of the key steps in the industrial production of recombinant proteins. In the current study, polydopamine nanoparticles (PDA-NPs) are considered in the synthesis of magnetic beads for purifying recombinant proteins due to advantages such as biocompatibility/ biodegradability, easy synthesis, as well as the ability to directly chelate metal ions. They were synthesized in Tris buffer (pH: 8:5), then chelated with Fe3+(20 mg) and Ni2+ ions at concentrations of 2, 3, 5, and 7 mg/ml. Prepared nanoparticles were characterized through scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), Inductively Coupled Plasma (ICP), and vibrating sample magnetometer (VSM). The size distribution of the particles was reported in the narrow range of 120–140 nm and 200 to 220 nm by the SEM image and DLS analysis, respectively. The chelation of ions on the surface of the nanoparticle was confirmed by the ICP technique with a magnetization of 35.42 emu/g. The highest adsorption rate of Ni2+ ions to polydopamine was obtained at a ratio of 1.4. The SDS-PAGE and western blot analysis confirmed the purification of eGFP and Hsp40 by PDA/Fe3+/Ni2+ at 26 and 40 kDa compared to the commercial nickel column. Moreover, the concentration of purified eGFP by PDA/Fe3+/Ni2+ was reported 138.83 µg/ml by the fluorescent signals, which is almost equal to or more than the protein purified by commercial Ni-NTA column (108.28 µg/ ml). The stability of PDA/Fe3+/Ni2+ has also been evaluated by ICP-OES for 10 days, and the result suggested that PDA magnetic beads were stable. Therefore, it can be concluded that PDA/Fe3+/Ni2+ have the ability to purify recombinant proteins in one less step and shorter time.

N-Acetylcysteine Displaces Glutathionyl-Moieties from Hg2+ and MeHg+ to Form More Hydrophobic Complexes at Near-Physiological Conditions.

The anthropogenic release of Hg is associated with an increased human exposure risk. Since Hg2+ and MeHg+ have a high affinity for thiols, their interaction with L-glutathione (GSH) within mammalian cells is fundamentally involved in their toxicological chemistry and excretion. To gain insight into the interaction of these mercurials with multiple small molecular weight thiols, we have investigated their competitive interactions with GSH and N-acetylcysteine (NAC) at near-physiological conditions, using a liquid chromatographic approach. This approach involved the injection of each mercurial onto a reversed-phase (RP)-HPLC column (37 °C) using a PBS buffer mobile phase containing 5.0 mM GSH to simulate cytosolic conditions with Hg being detected in the column effluent by an inductively coupled plasma atomic emission spectrometer (ICP-AES). When the 5.0 mM GSH mobile phase was amended with up to 10 mM NAC, gradually increasing retention times of both mercurials were observed. To explain this behavior, the experiment with 5.0 mM NAC and 5.0 mM GSH was replicated using 50 mM Tris buffer (pH 7.4), and the Hg-containing fractions were analyzed by electrospray ionization mass spectrometry. The results revealed the presence of Hg(GS)(NAC) and Hg(NAC)2 for Hg2+ and MeHg(GS) and MeHg(NAC) for MeHg+, which suggests that the coordination/displacement of GS-moieties from each mercurial by the more hydrophobic NAC can explain their retention behavior. Since the biotransformations of both mercurials were observed at near-physiological conditions, they are of toxicological relevance as they provide a biomolecular explanation for some results that were obtained when animals were administered with each mercurial and NAC.

Green and white MEKC for determination of different anti-diabetic binary mixtures and their triple-combo pill.

The work introduces green and white sustainable micellar electrokinetic chromatography (MEKC) procedure that could analyze therapeutically related drugs, empagliflozin (EMP), linagliptin (LIN) and metformin (MET) which are antidiabetic drugs with different mechanism of action, in their different pharmaceutical combinations. The method not only comply with the green analytical concepts, but also it is in line with sustainable analytical concepts as it is economic by applying the same operating conditions to analyze different pharmaceuticals in quality control (QC) labs which is a crucial step in QC labs and research centers to save time, effort, and money. Moreover, the method functionality regarding its scope with its achieved levels of accuracy, precision, low detection, and quantitation limits is tested using white assessment tool and compared with reported methods. The proposed MEKC coupled with a diode array detector (DAD) has been developed and validated for micro estimation of EMP and LIN in their low critical concentrations with MET in a ratio of (EMP: MET, 1:40) and (LIN: MET, 1:200). Separation was achieved within 6min using fused silica capillary (40cm × 50µm id) using 20mM Tris buffer (pH 10) in presence of 50mM sodium dodecyl sulphate and 10% v/v methanol. The concentration ranges of the studied anti-diabetic drugs were 10-500, 10-100 and 2.5-100µg. mL-1 for MET, EMP and LIN, respectively. The developed method is the first MEKC for concurrent determination of EMP, LIN and MET with high separation efficiency, low solvent consumption and regard as an easy green and white analytical tool. Moreover, Greenness and whiteness assessment were done via the most widely used Analytical Eco-Scale, the innovative AGREE tool and the RGB 12 algorithm.

Electrochemical Disposable Biosensor to Monitor Dabigatran in Point-of-Care Anticoagulation Therapy.

Dabigatran etexilate, an oral prodrug, is often used to treat complications linked to thrombosis. Dabigatran (DAB, active form) does not need to be monitored. However, there are several conditions, such as reduced renal function, traumatic bleeding, emergency surgery, the need for thrombolytic therapy in acute stroke, or the requirement to use other forms of anticoagulation, where knowing the concentration of DAB in the blood is indispensable. Unfortunately, there are no convenient DAB-specific point-of-care tests available. To solve this problem, two disposable sensors were constructed and optimised in this work to detect the anticoagulant drug DAB using novel co-facing disposable electrodes, which allows a calibration-free quantitation of the electroactive mediator concentration. A trypsin-based sensor was evaluated. This sensor performed well in a 10 mM Tris buffer (pH 8.8) solution. However, trypsin was inhibited by alpha-1 antitrypsin when a plasma sample was introduced into the sensor. This problem was overcome by plasma filtration. This sensor showed a detection limit of 50.7 ng mL-1 DAB in plasma and a quantification range of 177-500 ng mL-1. A thrombin-based sensor was also constructed. This sensor performed well in ten-fold diluted plasma, overcoming the filtration problem observed with the trypsin-based sensor. This sensor showed a detection limit of 9.6 ng mL-1 DAB in plasma and a quantification range of 11.5-140 ng mL-1. Its extensive pH stability range, the possibility of working at physiological pH, low volume, low cost, and fast turnaround response (less than 20 s) make the calibration-free thrombin-based sensor a suitable point-of-care test to measure DAB concentration in the blood.

2-Azahetaryl-2-(oxoindolin-2-ylidene)acetonitriles as Colorimetric Probes for Zn: Synthesis and Optical Properties.

A new one-pot approach for the synthesis of the Zn2+-sensitive probes 2-azahetaryl-2-(oxoindolin-2-ylidene)acetonitriles 3a-c and 4 is described. The method includes the in situ formation of imidoylchloride and its further condensation with azahetarylacetonitrile 1. The structure of the obtained compounds is studied using 1H nuclear magnetic resonance (NMR), 13C NMR, infrared (IR), high-resolution mass spectrometry (HRMS), and UV-Vis spectroscopy techniques. Two model ligands both exhibiting the highest extinction coefficient and the best solubility in a Tris buffer pH 7.2/dimethyl sulfoxide (DMSO) solution, namely 5-methyl-benzothiazole derivative 3b and benzoxazole derivative 4, are thoroughly studied as colorimetric probes for Zn2+. The probe 3b has the highest sensitivity to Zn2+, showing a limit of ion detection (LOD) calculated by the 3S criterion of 0.43 μM and selectivity upon masking Cu2+ ions with Na2S2O3. The composition of the complexes in the solution was determined by the limited logarithm method. The stability constant (lg K) values of 3b-Zn of 10.27 ± 0.02 and 4-Zn of 12.5 ± 0.2 indicate the formation of complexes of average stability.

Are inert glasses really inert?

The aim of this study was to investigate the degradation of inert glass fillers which are commonly used in conventional resin-based composites to provide radiopacity, reduce the polymerization shrinkage and improve the mechanical properties. 75mg of five different glass powders (1µm) was immersed separately into 50mL of acetic acid (pH 4) and tris buffer (pH 7.4) for up to 4 weeks. At each time point the glass powder was filtered and dried for characterization using ATR-FTIR and XRD to assess the degradation behavior and crystallization. ICP-OES, ISE and pH measurements were performed on the supernatant solutions to monitor the pH and ion release. Although FTIR and XRD analysis showed no significant glass degradation or crystallization upon immersion, there was a substantial release of ions from the inert fillers, especially from BABFG and CDL. Barium release for these fillers were 270 and 165ppm respectively. G018-373 glass presented the lowest ion release followed by GM27884 and BABG. The ion release was more pronounced in acidic conditions compared to neutral conditions apart from the fluoride release. Inert glasses are not as inert as previously thought. This may result in leaching of ions, potentially causing toxicity, reduction in mechanical properties, increased wear and subsequent failure of the composite material. The ions released from the inert glass may interfere with other glass fillers such as bioactive glass fillers, inhibiting degradation of the bioactive glass, beneficial ion release from the bioactive glass, pH neutralization and apatite formation.

Multifunctional chitosan/tannic acid composite films with improved anti-UV, antioxidant, and antimicrobial properties for active food packaging

Chitosan (CS) has been extensively studied because of its biocompatibility, biodegradability, low toxicity, and good film-forming properties. However, pristine CS has relatively low UV resistance, antioxidant, and antibacterial properties, and is thus not suitable for food packaging. In this study, multifunctional tannic acid (TA) was incorporated into pristine CS to overcome its limitations. Two neutralization conditions with different pH (pH 7.4 using phosphate-buffered saline (PBS) and pH 8.5 using Tris buffer) were employed to prepare CS-TA composite films. The chemical and structural characteristics of the composite films were analyzed through Fourier-transform infrared, UV–visible, and X-ray photoelectron spectroscopies. At the higher neutralization pH of 8.5, the covalent crosslinking between CS and TA increased because more TA was transformed into quinone form. In addition, the Schiff base reaction was predominant when neutralized with PBS (pH 7.4), whereas the Michael addition reaction was predominant when neutralized with Tris buffer (pH 8.5). The CS-TA composite films exhibited better mechanical, thermal, antioxidant, and antibacterial properties than those of pristine CS. Furthermore, the packaging film of the composite effectively reduced enzymatic browning and water loss from bananas. The findings suggest that the CS-TA composite films can be used as active packaging materials for long-term food storage owing to their excellent mechanical, antioxidant, and antibacterial properties.

Efficient side-chain deacylation of polymyxin B1 in recombinant Streptomyces strains.

Polymyxins are antibacterial polypeptides used as "last resort"therapy option for multidrug-resistant Gram-negative bacteria. The expansion of polymyxin-resistant infections has inspired development of novel polymyxin derivatives, and deacylation is one of the critical steps in generating those antibiotics. Deacylase from Actinoplanes utahensis hydrolyze the acyl moieties of echinocandins, and also efficiently deacylates daptomycin, ramoplanin and other important antibiotics. Here, deacylase was studied considering its potential usefulness in deacylating polymyxin B1. All the six recombinant strains containing the deacylase gene catalyzed hydrolysis of polymyxin B1, yielding cyclic heptapeptides. The efficiency of recombinant S. albus (SAL701) was higher than that of the others, and deacylation was the most efficient at 40°C in 0.2M Tris buffer (pH 8.0) with 0.2M Mg2+. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0g/L. SAL701 was highly thermostable, showing no loss of activity at 50°C for 12h, and the mycelia could be recycled at least three times without loss of catalytic activity. SAL701 could not deacylate β-lactam substrate such as penicillin G and cephalosporin C. Deacylase catalyzes the amide bond 1 closest to the nucleus of polymyxin B1 rather than the other bond, suggesting that it has high catalytic site specificity. Homology modeling and the docking results implied that Thr190 in deacylase could facilitate hydrolysis with high regioselectivity. These results show that SAL701 is effective in increasing the cyclic heptapeptide moiety of polymyxin B1. These properties of the biocatalyst may enable its development in the industrial production of polymyxins antibiotics.

Development of Heteroarene-Fused Quinones As Rapidly Dissoluble and Stable Biosensors

Aqueous electron mediators for point-of-care testing require a variety of properties, such as rapid dissolubility, high stability, and moderate formal potential (near 0 V). Using the redox-active properties of quinone derivatives, various quinone-containing electron mediators have been developed for electrochemical sensors. However, most quinone derivatives exhibit very low solubility in aqueous solutions and instability in the presence of nucleophilic species. Herein, we report a strategic design for quinones incorporating sulfonated thioether and nitrogen-containing heteroarene moieties as solubilizing, stabilizing, and formal potential-modulating groups. Systematic investigations found that di(thioether sulfonate)-substituted quinoline-1,4-dione (QLS) and quinoxaline-1,4-dione (QXS) displayed high water solubility and rapid dissolubility. By finely balancing the electron-donating effect of the thioethers and the electron-withdrawing effect of the nitrogen atom, formal potentials suitable for electrochemical biosensors are achieved with QLS and QXS (−0.15 and −0.09 V vs Ag/AgCl, respectively, at pH 7.4). QLS is stable for >1 d in PBS (pH 7.4) and for 1 h in tris buffer (pH 9.0), which is sufficient for point-of-care testing. Furthermore, QLS, with its high electron mediation ability, is successfully used in biosensors for sensitive detection of glucose and parathyroid hormone. This strategy provides organic electron mediators that exhibit rapid dissolution and high stability and will find broad applications beyond quinone-based biosensors. Figure 1

Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.

Synthesis of 225Ac-PSMA-617 for Preclinical Use.

The recent approval of radiopharmaceuticals for diagnosis and treatment of cancer is ushering nuclear medicine into a new era of theranostics and alpha therapy using radiopharmaceuticals labeled with 225Ac shows remarkable results in clinical trials. As such, reliable methods for the synthesis and quality control of 225Ac-radiopharmaceuticals are needed. 225Ac-PSMA-617 is being used for targeted alpha therapy in patients with prostate cancer, and we had cause to synthesize the agent for preclinical use. However, technology transfer proved cumbersome owing to the paucity of information available on synthesizing and analyzing 225Ac-radiotherapeutics. To address this need, we describe a straightforward synthesis of 225Ac-PSMA- 617 as well as suitable approaches for quality control analysis using standard equipment in a modern PET Center. PSMA-617 precursor was dissolved in 25 μL metal-free water (0.67 mg/mL) and combined with 500 μL 0.05M Tris buffer, pH 9. Actinium stock solution (~65 μCi in 15 μL) was added and the reaction was heated at 120°C for 40-50 min. The reaction was cooled and 0.6 mL gentisic acid solution (4 mg/mL in 0.2 M NH4OAc) was added. To formulate the dose for injection, sterile saline, USP (8 mL) was added and the pH was adjusted by the addition of 100 μL 0.05 M Tris buffer (pH 9) to give a final pH of ~7.2. The final solution was filtered using a 0.22 μm GV sterile filter into a sterile dose vial. Radiochemical purity was determined by radio-TLC (eluent: 50mM Sodium Citrate, pH 5), and plates were analyzed using an AR2000 scanner. The method provided 225Ac-PSMA-617 in high radiochemical yield (57 ± 3 μCi, >99%) and radiochemical purity (98 ± 1%), formulated for preclinical studies (9 mL, pH = 7.2), n=3. A straightforward synthesis of 225Ac-PSMA-617 is described that will facilitate production for (pre)clinical studies. The approach could also be applicable to the synthesis of other alpha radiotherapeutics incorporating 225Ac.

Phenolics, proteins, antioxidant activity and GC – MS analysis of Artocarpus heterophyllus Lam seeds

Artocarpus heterophyllus seeds are available in larger quantity and are good source for many phytochemicals. The seeds are rich in proteins and low in fat content. The present study was carried out to analyse the phenolics, proteins and antioxidant activity of A. heterophyllus seeds. Different aqueous solvents such as acetate buffer pH 5.0, phosphate buffer pH 7.0, tris buffer pH 8.5, different concentration of sodium hydroxide (0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M), sodium chloride, water and organic solvents like ethanol, methanol and acetone were used for extraction. Sodium hydroxide (0.1M) showed maximum extraction efficiency. Among the organic solvents, ethanol showed maximum phytochemical extraction. Different concentrations of ethanol (20%, 40%, 60%, 80%) containing 0.1M NaOH were also used for extraction and maximum extraction was found in 40% ethanol containing 0.1M NaOH. GC – MS analysis was carried to identify various bioactive compounds present in the extracts (hexane, ethyl acetate, methanol and water) of the seeds.

A method for purifying nanoparticles using cationic modified monoliths and aqueous elution

Nanoparticles are widely used in the medical field for diagnosis and therapy. In particular, the use of nanoparticles containing vaccines has spread rapidly; hence, ensuring nanoparticle safety and minimizing their side effects have become important concerns worldwide. In this study, we used three types (NH2, poly-Lys, and trimethylaminopropyl) of cationic modified silica monoliths with cylindrical structures, diameters of 4.2 mm, and heights of 1.5 mm. Doxil, an anticancer nanomedicine, and exosomes, as typical nanoparticles, were separated from model leaked drugs (e.g., doxorubicin and oligonucleotides) and proteins (e.g., albumin) coexisting in nanoparticle sample solutions using these monoliths. Each nanoparticle solution (200 μL) was applied to each monolith followed by centrifugation at 9,100 g for 1 min. The ionic concentration of the elution solution was increased stepwise to determine the concentration required to elute the nanoparticles from each monolith by centrifugation. The NH2- and poly-Lys-modified monoliths separated and purified nanoparticles from leaked drugs or proteins coexisting in nanoparticle sample solutions. The nanoparticles were separated from other substances by changing the pH and concentration of the aqueous Tris buffer used as the eluent. Doxil was eluted with 500–1,000 mM Tris buffer (pH 8) when using the NH2-modified monolith, and with 200–1,000 mM Tris buffer (pH 6) when using the poly-Lys-modified monolith. Exosome was obtained using 1,000 mM Tris buffer (pH 8) and the NH2-modified monolith. The recovery efficiencies (ratio of nanoparticle content in the most abundant fraction to that in the sample solution before purification) of Doxil and exosome were 64% and 55%, respectively. Because this method can purify nanoparticles using only low-speed centrifugation for a few minutes, we expect it will be used to improve nanoparticle safety.

Evaluation of intermolecular interactions required for thermostability of a recombinant adenovirus within a film matrix

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7–1.17 × 10−4 M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10−9 M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process.

Di(Thioether Sulfonate)-Substituted Quinolinedione as a Rapidly Dissoluble and Stable Electron Mediator and Its Application in Sensitive Biosensors.

The commonly required properties of diffusive electron mediators for point-of-care testing are rapid dissolubility, high stability, and moderate formal potential in aqueous solutions. Inspired by nature, various quinone-containing electron mediators have been developed; however, satisfying all these requirements remains a challenge. Herein, a strategic design toward quinones incorporating sulfonated thioether and nitrogen-containing heteroarene moieties as solubilizing, stabilizing, and formal potential-modulating groups is reported. A systematic investigation reveals that di(thioether sulfonate)-substituted quinoline-1,4-dione (QLS) and quinoxaline-1,4-dione (QXS) display water solubilities of ≈1 m and are rapidly dissoluble. By finely balancing the electron-donating effect of the thioethers and the electron-withdrawing effect of the nitrogen atom, formal potentials suitable for electrochemical biosensors are achieved with QLS and QXS (-0.15 and -0.09V vs Ag/AgCl, respectively, at pH 7.4). QLS is stable for >1d in PBS (pH 7.4) and for 1 h in tris buffer (pH 9.0), which is sufficient for point-of-care testing. Furthermore, QLS, with its high electron mediation ability, is successfully used in biosensors for sensitive detection of glucose and parathyroid hormone, demonstrating detection limits of ≈0.3× 10-3 m and ≈2 pg mL-1 , respectively. This strategy produces organic electron mediators exhibiting rapid dissolution and high stability, and will find broad application beyond quinone-based biosensors.