A self-heating cartridge for molecular diagnostics

A disposable, water-activated, self-heating, easy-to-use, polymeric cartridge for isothermal nucleic acid amplification and visual fluorescent detection of the amplification products is described. The device is self-contained and does not require any special instruments to operate. The cartridge integrates chemical, water-triggered, exothermic heating with temperature regulation facilitated with a phase-change material (PCM) and isothermal nucleic acid amplification. The water flows into the exothermic reactor by wicking through a porous paper. The porous paper's characteristics control the rate of water supply, which in turn controls the rate of exothermic reaction. The PCM material enables the cartridge to maintain a desired temperature independent of ambient temperatures in the range between 20 °C and 40 °C. The utility of the cartridge is demonstrated by amplifying and detecting Escherichia coli DNA with loop mediated isothermal amplification (LAMP). The device can detect consistently as few as 10 target molecules in the sample. With proper modifications, the cartridge also can work with other isothermal nucleic acid amplification technologies for detecting nucleic acids associated with various pathogens borne in blood, saliva, urine, and other body fluids as well as in water and food. The device is suitable for use at home, in the field, and in poor-resource settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.     

Molecular modeling of Protein A affinity chromatography

The properties of the complex between fragment B of Protein A and the Fc domain of IgG were investigated adopting molecular dynamics with the intent of providing useful insight that might be exploited to design mimetic ligands with properties similar to those of Protein A. Simulations were performed both for the complex in solution and supported on an agarose surface, which was modeled as an entangled structure constituted by two agarose double chains. The energetic analysis was performed by means of the molecular mechanics Poisson Boltzmann surface area (MM/PBSA), molecular mechanics generalized Born surface area (MM/GBSA), and the linear interaction energy (LIE) approaches. An alanine scan was performed to determine the relative contribution of Protein A key amino acids to the complex interaction energy. It was found that three amino acids play a dominant role: Gln 129, Phe 132 and Lys 154, though also four other residues, Tyr 133, Leu 136, Glu 143 and Gln 151 contribute significantly to the overall binding energy. A successive molecular dynamics analysis of Protein A re-organization performed when it is not in complex with IgG has however shown that Phe 132 and Tyr 133 interact among themselves establishing a significant π–π interaction, which is disrupted upon formation of the complex with IgG and thus reduces consistently their contribution to the protein–antibody bond. The effect that adsorbing fragment B of Protein A on an agarose support has on the stability of the protein–antibody bond was investigated using a minimal molecular model and compared to a similar study performed for a synthetic ligand. It was found that the interaction with the surface does not hinder significantly the capability of Protein A to interact with IgG, while it is crucial for the synthetic ligand. These results indicate that ligand–surface interactions should be considered in the design of new synthetic affinity ligands in order to achieve results comparable to those of Protein A right from the ligand design stage.     

New affinity nylon membrane used for adsorption of gamma-globulin

Microporous polyamide membranes were first modified by acid hydrolysis and subsequently bound with hydroxy-ethylcellulose to amplify reactive groups and reduce nonspecific interactions with proteins. Then 1,6-diaminohexane as space arm and phenylalanine as ligand were immobilized onto the nylon membranes by s-triazine trichloride activation. Affinity membranes thus obtained were set in a stack and used to adsorb gamma-globulin. The adsorption capacity (qm) of the affinity membrane is 53 micro gamma-globulin per m2 membrane and the desorption constant (Kd) is 2.35 x 10(-6) mol/l. The effects of feed, washing and elution rates on adsorption and desorption behavior were investigated. The results showed that affinity purification through these membranes could not be operated at very high flow-rates. A stack of 20 membranes with 47 mm diameter can adsorb 7.8 mg gamma-globulin with a purity of 91.6% from 4 ml of human plasma in a single-pass mode.     

New low-cost tubular ceramic microfiltration membrane based on natural sand for tangential urban wastewater treatment

In wastewater treatment, the development of low-cost separation methods is of significant importance. Low-cost membranes based on natural materials have become a highly active research topic in recent years. Herein, using low-cost natural Moroccan sand, new ceramic supports have been developed and characterized using different techniques such as X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), differential thermal analysis (DTA), along with scanning electron microscope (SEM). Plastic paste (average particle size ≤125 µm) was blended with organic additives and water, then the obtained paste was extruded into porous tubular supports. The support had a porosity of 43%, water permeability of 1928 L/h m² bar, excellent chemical and mechanical properties and an average pore diameter in the range of 8–15 µm after firing at 950 °C/2 h. As per SEM analysis, the tubular supports had a smooth and crack-free surface. The slip casting process was used to create a microfiltration layer from the same natural sand powder (average particle size ≤63 µm) using a mixture of powder sand, water, and polyvinyl alcohol solution. The water permeability of the microfiltration membrane sintered at 950 °C/2 h was 1052 L/h m² bar, the average pore size diameter was about 0.90 µm and 82% of pores had a diameter ≤1.00 µm. The obtained microfiltration membrane was tested for the treatment of urban wastewater. The membrane showed excellent separation performance in turbidity removal and chemical oxygen demand.     

Hybrid microfluidic cartridge formed by irreversible bonding of SU-8 and PDMS for multi-layer flow applications

SU-8 and polydimethylsiloxane (PDMS) are both transparent materials with properties very convenient for rapid prototyping of microfluidic systems. However, previous efforts of combining these two materials failed due to poor adhesion between them. Herein, we introduce a promising low-temperature technique (< 100 °C) to irreversibly bond two or more structured layers of SU-8 and PDMS to create hybrid stacks. This offers new possibilities in design and fabrication of enclosed three-dimensional microstructures and microchannels with simple soft-lithography techniques. The potential of this method is demonstrated by the fabrication of a new version of our microfluidic sensor cartridge that was reported recently¹.     

Protein ligand docking based on empirical method for binding affinity estimation

An empirical protein-ligand binding affinity estimation method, SCORE, was incorporated into a popular docking program, DOCK4. The combined program, ScoreDock, was used to reconstruct the 200 protein-ligand complex structures and found to give good results for the complexes with high binding affinities. A quality assessment method for docking results from ScoreDock was developed based on the whole test set and tested by additionally selected complexes. The method significantly improves the docking accuracy and was shown to be reliable in docking quality assessment. As a docking tool in structural based drug design, ScoreDock can screen out final hits directly based on the predicted negative logarithms of dissociation equilibrium constants of protein-ligand complexes, and can explicitly deal with structure water molecules, as well as metal atoms.     

IDA型Cu~(2+):-螯合亲和膜色谱法对牛肝过氧化氢酶纯化的研究(英文):

首次采用以改性纤维素为基质、亚氨二乙酸（ＩＤＡ）为取代配基的铜离子螯合膜色谱法对牛肝过氧化氢酶（ＢＬＣ）的分离纯化进行了研究。缓冲液的ｐＨ值对ＢＬＣ与螯合配基的结合影响显著。在选定的色谱条件下,ＢＬＣ粗酶液经ＩＤＡ型Ｃｕ２＋－螯合膜色谱柱一步纯化,比活性平均提高４．７倍,回收率为６７．７％。金属螯合膜色谱柱可用含０．２ｍｏｌ／Ｌ的咪唑或５０ｍｍｏｌ／ＬＥＤＴＡ－１ｍｏｌ／ＬＮａＣｌ的缓冲液再生,反复使用,后者比前者对柱子的再生效果更好。     

Enhanced binding by dextran‐grafting to Protein A affinity chromatographic media

Dextran‐grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose‐based matrix, followed by epoxy‐activation and Protein A coupling site‐directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran‐grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction‐dried gel, increased by 24% compared with that of the non‐grafted medium. The binding capacity of dextran‐grafted medium decreased about 7% after 40 cleaning‐in‐place cycles, much lower than that of the non‐grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran‐grafted medium faster than that of non‐grafted one. Atomic force microscopy showed that this dextran‐grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non‐grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high‐performance dextran‐grafted Protein A affinity chromatographic medium has promising applications in large‐scale antibody purification.     

Comparative study of reaction kinetics in membrane and agarose bead affinity systems.

Compared to conventional agarose bead affinity supports, a microporous nylon membrane exhibits greatly improved reaction kinetics as quantified in the reaction between gamma-globulin and immobilised protein A. The improvement is only observed when the solution of gamma-globulin is forced through the membrane pores. In the absence of flow in the pores, it is possible to relate approximately the rate of uptake onto either type of affinity support to independently determined diffusion coefficients. In the presence of flow, the reaction rate is similar for membranes having 0.45 and 3.0 microns diameter pores, and considerably smaller than predicted by the Smoluchowski formula.     

Immobilized metal affinity composite membrane based on cellulose for separation of biopolymers

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Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K_d values) and non-linear chromatography can be used to assess the association (k_on) and dissociation (k_off) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.     

Elaboration,characterization and study of a new hybrid chitosan/ceramic membrane for affinity membrane chromatography

A new kind of metal affinity membrane based on a ceramic support was prepared. It was elaborated in four steps: (i) deposition of a chitosan layer in order to functionalize the ceramic support, (ii) cross-linking with epichlorohydrin to stabilise the polymer layer and to enable the grafting, (iii) iminodiacetic acid grafting, (iv) Cu²⁺ adsorption. Due to the ceramic support, this membrane is highly resistant and the chitosan layer brings its biocompatibility properties. Each step of the membrane elaboration was studied and the membrane structure was characterized. Both thin coating of the polymer on the alumina grains of the support and the chemical modification of the membrane were proved. Then, bovine serum albumin (BSA) was used as a model protein to test protein retention of the affinity membrane. The protein/membrane interactions were investigated showing that some non-specific ones are involved. Finally, the effect of buffer concentration was checked and it appears that, in the studied range, an increase of the buffer concentration entailed a limitation of the non-specific interactions inducing a better BSA recovering and a higher selectivity.     

Biological membrane derived nanomedicines for cancer therapy

The development of nanomedicine systems for applications in cancer therapies has been widely explored in the last decade. With inherent biocompatibility, nanomedicine devices derived from biological membranes have shown many unique advantages compared with traditional artificial nanomaterials for biomedical applications. Herein, we present a comprehensive review of the recent development of cell membrane derived nanomedicines in cancer treatment. We firstly outline the advantages of biological membranes in nanomedicine design derived from their intrinsic characteristics, and then discuss the applications of biological membrane derived nanomedicines. For the first major category of membrane-derived nanomedicine, synthetic nanoparticles are usually camouflaged with cell membranes to acquire additional functionalities. The other type of membrane-based nanomedicine is directly using the engineered cell membrane-derived vesicles or nanovesicles secreted by cells for tumor treatment. At last, we discuss the challenges of membrane-derived nanomedicines towards future clinical applications, following with perspectives on possible solutions to the current problems.     

A highly stable membrane with hierarchical structure for wide pH range flow batteries

A membrane with high stability and ion conductivity in wide pH range is essential for energy storage devices.Here,we report a novel membrane with hierarchical core-shell structure,which demonstrates high stability and ion conductivity,simultaneously under a wide pH range applications.Spectral characterizations and theoretical calculation indicate that the non-solvent induces the chain segment configuration and eventually leads to polymer-polymer phase separation,thus forming hierarchical porous core-shell structure.Benefiting from this structure,an acidic vanadium flow battery(VFB)with such a membrane shows excellent performance over 400 cycles with an energy efficiency(EE)of above 81%at current density of 120 mA cm^-2 and an alkaline zinc-iron flow battery(AZIFB)delivers a cycling stability for more than 200 cycles at 160 mA cm^-2,along with an EE of above 82%.This paper provides a cost-effective and simple way to fabricate membranes with high performance for variety of energyrelated devices.     

A flow cell optosensor for lead based on immobilized gallocynin in chitosan membrane

Gallocynin immobilized in chitosan membrane has been studied as a sensor element of an optical sensor for lead using a flowing system. By using this set up, lead in solution has been determined in the concentration range from 1.0x10(-1) to 1.0x10(3) ppm with a detection limit of 0.075 ppm. The standard deviation of the method for the repeatability of lead detection at a concentration of 100 ppm was found to be 2.10%. The response of the sensor was reproducible and can be regenerated by using acidified saturated KNO(3) solution. Interference from foreign ions was also studied at 1:1 mole ratio of Pb(II):foreign ions.