Agarose Materials Research Articles

Hierarchical Macroporous Agarose Materials with Polyethyleneimine-Assisted Multiple Boronate Affinity Binding Sites for the Separation of Neomycin.

Quantification of neomycin residues in food samples demands an efficient purification platform. Herein, hierarchical macroporous agarose monoliths with multiple boronate affinity sites were established for selective separation of neomycin. The silica core was synthesized by "one-step" Stöber procedures followed by modification with amino group and incorporation of polyethyleneimine. A versatile macroporous agarose monolith was prepared by emulsification strategies and functionalized with epoxy groups. After introducing polyethyleneimine-integrated silica nanoparticles onto the agarose monolith, fluorophenylboronic acids were immobilized. The physical and chemical characteristics of the composite monolith were analyzed systematically. After optimization, neomycin showed high binding ability of 23.69 mg/g, and the binding capacity can be manipulated by changing the pH and adding monosaccharides. The composite monolith was subsequently utilized to purify neomycin from the spiked model aquatic products followed by high-performance liquid chromatography analysis, which revealed a remarkable neomycin purification effect, indicating the great potential in the separation of neomycin from complicated aquatic products.

PRECLINICAL EVALUATION OF THIN CONVEX PROBE ENDOBRONCHIAL ULTRASOUND-GUIDED TRANSBRONCHIAL NEEDLE ASPIRATION FOR INTRAPULMONARY LESIONS

TOPIC: Procedures TYPE: Original Investigations PURPOSE: A prototype thin convex probe endobronchial ultrasound scope (TCP-EBUS; distal outer diameter [OD] 5.9 mm; Olympus Medical Systems Corp., Japan) was developed to enable access to distal airways and diagnose intrapulmonary lesions. The objective of this study was to evaluate the potential clinical utility of TCP-EBUS by preclinically assessing its bronchial accessibility, the feasibility of EBUS-guided transbronchial needle aspiration (EBUS-TBNA) of intrapulmonary targets, as well as its short- and medium-term safety. METHODS: For bronchial accessibility, the TCP-EBUS was inserted into the distal airways of ex-vivo human lungs (n=5). TCP-EBUS was compared with several clinically-available bronchoscopes (BF-H190 bronchoscope, OD 5.5 mm; BF-Q190 bronchoscope, OD 4.8 mm; BF-Y0063 CP-EBUS scope, OD 6.6 mm, Olympus Medical Systems Corp., Japan). The maximum reach, in terms of bronchial generation, for each scope was documented using fluoroscopy and bronchoscopy. To quantify the minimum diameter of the distal airway accessed by the TCP-EBUS, an agarose-barium sulfate mixture was injected transbronchially to mark the bronchus where the TCP-EBUS could reach, followed by cone-beam CT to measure the diameter radiographically (n=3). For intrapulmonary lesion EBUS-TBNA feasibility, an agarose-barium mixture was injected transbronchially into the lungs in a live pig to create pseudo-tumor targets; successful EBUS-TBNA was based on retrieval of dyed agarose material. The short- (≤1 hr) and medium-term (≤10 d) safety of TCP-EBUS insertion and EBUS-TBNA in the periphery were observed physiologically and radiologically (fluoroscopy, computed tomography [CT]) in live pigs (n=5). RESULTS: For bronchial accessibility, TCP-EBUS demonstrated greater reach than the standard CP-EBUS in all bronchi, as well as surpassed the 5.5 mm-bronchoscope in 62.7% (131/209) and the 4.8 mm-bronchoscope in 27.2% (57/209) of assessed bronchi. The median bronchial generation and the mean diameter of bronchi TCP-EBUS reached was 4 (range 3-7) and 3.3±0.7 mm, respectively. No difficulties with EBUS-TBNA of intrapulmonary pseudo-tumors were observed. No major complications related to TCP-EBUS-TBNA in distal airways were observed in the five pigs. Scattered mucosal erythema of the bronchial walls was observed immediately after TCP-EBUS insertion in all pigs; this self-resolved by day 10. One pig developed an inflammatory scar in the right upper lobe by day 10. CONCLUSIONS: TCP-EBUS insertion appears to be safe and improves access to the distal airways. It has the potential to facilitate EBUS-TBNA of intrapulmonary lesions in the central and middle lung fields. CLINICAL IMPLICATIONS: Ultrasound-guided real-time sampling for intrapulmonary lesions located in the inner two-thirds lung fields may be safely conducted with the TCP-EBUS bronchoscope. DISCLOSURES: No relevant relationships by Masato Aragaki, source=Web Response No relevant relationships by Nicholas Bernards, source=Web Response No relevant relationships by Harley Chan, source=Web Response No relevant relationships by Zhenchian Chen, source=Admin input No relevant relationships by Andrew Effat, source=Web Response No relevant relationships by Alexander Gregor, source=Web Response No relevant relationships by Terunaga Inage, source=Web Response No relevant relationships by TSUKASA ISHIWATA, source=Web Response No relevant relationships by Tomonari Kinoshita, source=Web Response No relevant relationships by Yamato Motooka, source=Web Response no disclosure on file for Koichiro Tatsumi; No relevant relationships by Hideki Ujiie, source=Web Responseresearch support relationship with Olympus Corporation Please note: $5001 - $20000 by Kazuhiro Yasufuku, source=Web Response, value=Grant/Research Consultant relationship with Olympus America Inc Please note: $1001 - $5000 by Kazuhiro Yasufuku, source=Web Response, value=Consulting fee Consultant relationship with Medtronic Please note: $1001 - $5000 by Kazuhiro Yasufuku, source=Web Response, value=Consulting fee Consultant relationship with Johnson and Johnson Please note: $1001 - $5000 by Kazuhiro Yasufuku, source=Web Response, value=Consulting fee Consultant relationship with Auris Surgical Robotics Please note: $1001 - $5000 by Kazuhiro Yasufuku, source=Web Response, value=Consulting fee

Architecture-Promoted Biomechanical Performance-Tuning of Tissue-Engineered Constructs for Biological Intervertebral Disc Replacement.

Background: Biological approaches to intervertebral disc (IVD) restoration and/or regeneration have become of increasing interest. However, the IVD comprises a viscoelastic system whose biological replacement remains challenging. The present study sought to design load-sharing two-component model systems of circular, nested, concentric elements reflecting the nucleus pulposus and annulus fibrosus. Specifically, we wanted to investigate the effect of architectural design variations on (1) model system failure loads when testing the individual materials either separately or homogeneously mixed, and (2) also evaluate the potential of modulating other mechanical properties of the model systems. Methods: Two sets of softer and harder biomaterials, 0.5% and 5% agarose vs. 0.5% agarose and gelatin, were used for fabrication. Architectural design variations were realized by varying ring geometries and amounts while keeping the material composition across designs comparable. Results: Variations in the architectural design, such as lamellar width, number, and order, combined with choosing specific biomaterial properties, strongly influenced the biomechanical performance of IVD constructs. Biomechanical characterization revealed that the single most important parameter, in which the model systems vastly exceeded those of the individual materials, was failure load. The model system failure loads were 32.21- and 84.11-fold higher than those of the agarose materials and 55.03- and 2.14-fold higher than those of the agarose and gelatin materials used for system fabrication. The compressive strength, dynamic stiffness, and viscoelasticity of the model systems were always in the range of the individual materials. Conclusions: Relevant architecture-promoted biomechanical performance-tuning of tissue-engineered constructs for biological IVD replacement can be realized by slight modifications in the design of constructs while preserving the materials’ compositions. Minimal variations in the architectural design can be used to precisely control structure–function relations for IVD constructs rather than choosing different materials. These fundamental findings have important implications for efficient tissue-engineering of IVDs and other load-bearing tissues, as potential implants need to withstand high in situ loads.

A smart microfluidic-based fish farm for zebrafish screening

Advances in microfluidics for zebrafish processing are critical to facilitate the bioassay in new drug development. Still, a manipulation platform for zebrafish relies mainly on the “static” agarose material and is coupled with the conventional fish facility to ensure the time-history investigation with the homogenous zebrafish population for drug screening. A fully automated IoT (Internet of Things) microfluidics system is necessary to be developed to enable smart and high-speed zebrafish testing without the need for fish facility support. In this work, a smart microfluidic device was presented to enable (i) highly efficient temperature control with the precision of ± 0.1 °C, (ii) remote zebrafish transport through light patterning, and (iii) perfusion and dynamic culturing of zebrafish all together assembled for on-chip high-quality imaging. A new microscale manipulation is envisaged to initiate a new chapter of running bioassays.

Determinants of protein elution rates from preparative ion-exchange adsorbents

The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor

A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1h at 60°C) when compared to PAMO in solution (no activity detected after 1h at 60°C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.

Acoustic radiation force contrast in MRI: Detection of calcifications in tissue‐mimicking phantoms

Mammography is a widely used tool for the screening of breast cancer, and calcifications are a common finding in most mammograms. The location, size, number, morphology, and distribution of calcifications are an important information to differentiate a benign lesion from probably malignant pathologies. Calcifications are not detectable with a standard dynamic contrast enhanced breast MRI. The authors present a novel method for the detection and imaging of calcifications in breast tissue without ionizing radiation or contrast agents. Measurements of localized tissue displacement in phantoms due to applied acoustic radiation force were performed. This displacement was imaged with a displacement sensitive spin-echo MRI sequence. Pieces of eggshell that represent calcifications were embedded in tissue-mimicking agarose phantoms. The sizes of the calcifications were 0.8 x 0.8 x 0.4, 1.5 x 1.5 x 0.4, and 2 x 3 x 0.4 mm3. The calcifications were scanned with ultrasound (U.S.) at 2.5 MHz and intensities up to I(spta) =7.18 W/cm2. The U.S. beam was moved inside the phantom by a computer-controlled three-dimensional hydraulic positioning system. The U.S. beam was scanned over the two smaller calcifications with the displacement sensitivity of the MRI sequence parallel to the U.S. beam path. Grayscale coded maps of the displacement scans are presented. For the 0.8 x 0.8 x 0.4 mm3 calcification, the U.S. intensities were varied. Finite element simulations were performed to verify if the experiments complied with theory. The authors found that the displacement caused by the U.S. is increased at the position of the calcification. The area of increased displacement is at least twice as large as the calcification itself. The simulations show this increase in displacement and area at the position of the calcification. When changing the displacement sensitivity direction to perpendicular to the U.S. beam, a crossed black and white four-leaf clover is visible at the position of the calcification. The U.S. is scattered and reflected by the calcifications. This leads to the increased displacement which is transmitted to the surrounding material because of the elastic coupling between the calcification and the agarose material. Due to the high differences in acoustic impedance and elastic properties between the surrounding tissue and the calcification, even the detection of pieces smaller than the resolution of the MRI scanner is possible. The acoustic radiation force contrast in MR phase-difference images offers a positive signal for calcifications from a smooth background in phantoms. This method offers a possibility of differentiating qualitatively and quantitatively hard calcifications from stiffer inclusions such as tumors.

“Clickable” Agarose for Affinity Chromatography

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

Superporous agarose monoliths as mini-reactors in flow injection systems. On-line monitoring of metabolites and intracellular enzymes in microbial cultivation processes.

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15 x 5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 microm in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 A) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min(-1)). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular beta-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against beta-galactosidase).

Continuous superporous agarose beds for chromatography and electrophoresis

Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20–200 μm) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD + analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.

Superporous agarose, a new material for chromatography

This paper reports on a new type of spherical agarose chromatography particles characterized by two sets of pores, normal diffusion pores, characteristic of all agarose materials and very wide pores, so-called superpores or flow pores. These superpores allow part of the chromatographic flow to pass through each individual particle, which gives improved mass transfer, especially in situations where diffusion is the limiting factor for the overall performance of a chromatographic separation. The particles were prepared by a double emulsification procedure. Observations under a microscope and size-exclusion chromatography were used in order to demonstrate pore flow. The chromatographic behaviour of the new particles was as efficient as that of homogeneous particles which were several times smaller. The agarose particles were derivatized with polyethyleneimine and used for an ion-exchange chromatographic separation of three model proteins. As expected from a perfusion material, the superporous beads resolved the protein mixture more efficiently than homogeneous beads of the same size.

Purification of monoclonal antibodies from whole hybridoma fermentation broth by fluidized bed adsorption

To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. (c) 1995 John Wiley & Sons, Inc.

Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.

Advantages of agarose gel electrophoresis and immunofixation for characterizing immunoglobulin abnormalities in human serum

Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20–200 μm) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD+ analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.

Affinity chromatography systems for artificial liver support.

The physical, adsorptive, and flow properties of two albumin conjugated agarose materials have been investigated in vitro to determine whether they could be used in artificial liver support systems to enhance the removal of toxic protein-bound substances. The results of binding of chenodeoxycholic acid and unconjugated bilirubin and flow experiments with blood and saline suggested that there could be advantages in using the SepharoseR rather than Bio--GelR albumin agarose in future haemoperfusion systems. However, further modifications are needed before the former can be successfully scaled up for human use.