3D Extrusion Printing of Biphasic Anthropomorphic Brain Phantoms Mimicking MR Relaxation Times Based on Alginate-Agarose-Carrageenan Blends.

The availability of adapted phantoms mimicking different body parts is fundamental to establishing the stability and reliability of magnetic resonance imaging (MRI) methods. The primary purpose of such phantoms is the mimicking of physiologically relevant, contrast-creating relaxation times T1 and T2. For the head, frequently examined by MRI, an anthropomorphic design of brain phantoms would imply the discrimination of gray matter and white matter (WM) within defined, spatially distributed compartments. Multichannel extrusion printing allows the layer-by-layer fabrication of multiple pastelike materials in a spatially defined manner with a predefined shape. In this study, the advantages of this method are used to fabricate biphasic brain phantoms mimicking MR relaxation times and anthropomorphic geometry. The printable ink was based on purely naturally derived polymers: alginate as a calcium-cross-linkable gelling agent, agarose, ι-carrageenan, and GdCl3 in different concentrations (0-280 µmol kg-1) as the paramagnetic component. The suggested inks (e.g., 3Alg-1Agar-6Car) fulfilled the requirements of viscoelastic behavior and printability of large constructs (>150 mL). The microstructure and distribution of GdCl3 were assessed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX). In closely monitored steps of technological development and characterization, from monophasic and biphasic samples as printable inks and cross-linked gels, we describe the construction of large-scale phantom models whose relaxation times were characterized and checked for stability over time.

Diffusion mechanisms of DNA in agarose gels: NMR studies and Monte Carlo simulations.

We report on the diffusion mechanism of short, single-stranded DNA molecules with up to 100 nucleobases in agarose gels with concentrations of up to 2.0% with the aim to characterize the DNA-agarose interaction. The diffusion coefficients were measured directly, i.e., without any model assumptions, by pulsed field gradient nuclear magnetic resonance (PFG-NMR). We find that the diffusion coefficient decreases, as expected, with an increase in both DNA strand length and gel concentration. In addition, we performed Monte Carlo simulations of particle diffusion in a model network of polymer chains, considering our experimental conditions. Together, the Monte Carlo simulations and the PFG-NMR results show that the decrease in diffusion coefficients in the presence of the agarose gel is due to a temporary adhesion of the DNA molecules to the surface of gel fibers. The average adhesion time to a given gel fiber increases with the length of the DNA strands but is independent of the number of gel fibers. The corresponding magnitude of the binding enthalpies of DNA strands to gel fibers indicates that a mixture of van der Waals interactions and hydrogen bonding contributes to the decreased diffusion of DNA in agarose gels.

Seaweed Polysaccharide Based Products and Materials: An Assessment on Their Production from a Sustainability Point of View.

Among the various natural polymers, polysaccharides are one of the oldest biopolymers present on the Earth. They play a very crucial role in the survival of both animals and plants. Due to the presence of hydroxyl functional groups in most of the polysaccharides, it is easy to prepare their chemical derivatives. Several polysaccharide derivatives are widely used in a number of industrial applications. The polysaccharides such as cellulose, starch, chitosan, etc., have several applications but due to some distinguished characteristic properties, seaweed polysaccharides are preferred in a number of applications. This review covers published literature on the seaweed polysaccharides, their origin, and extraction from seaweeds, application, and chemical modification. Derivatization of the polysaccharides to impart new functionalities by chemical modification such as esterification, amidation, amination, C-N bond formation, sulphation, acetylation, phosphorylation, and graft copolymerization is discussed. The suitability of extraction of seaweed polysaccharides such as agar, carrageenan, and alginate using ionic solvent systems from a sustainability point of view and future prospects for efficient extraction and functionalization of seaweed polysaccharides is also included in this review article.

Development and assessment of a novel ex vivo corneal culture technique involving an agarose-based dome scaffold for use as a model of in vivo corneal wound healing in dogs and rabbits.

OBJECTIVE: To develop and assess a novel ex vivo corneal culture technique involving an agarose-based dome scaffold (ABDS) for use as a model of in vivo corneal wound healing in dogs and rabbits. SAMPLE: Corneas from clinically normal dogs (paired corneas from 8 dogs and 8 single corneas) and rabbits (21 single corneas). PROCEDURES: 8 single dog corneas (DCs), 1 DC from each pair, and 10 rabbit corneas (RCs) were wounded with an excimer laser; 1 DC from each pair and 11 RCs remained unwounded. Corneas were cultured for 21 days on ABDSs (8 pairs of DCs and all RCs) or on flat-topped scaffolds (8 single DCs). The surface area of corneal fluorescein retention was measured every 6 (DCs) or 12 (RCs) hours until full corneal epithelialization was detected. Changes in corneal clarity were evaluated at 0, 7, 14, and 21 days. RESULTS: Median time to full epithelialization for wounded dog and rabbit corneas was 48 and 60 hours, respectively; among wounded DCs, time to full epithelization did not differ by scaffold type. After 21 days of culture on ABDSs, all DCs and RCs that epithelialized developed a circular, diffuse, cloud-like pattern of optical haze, whereas DCs cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze. All corneas on the ABDSs maintained convex curvature throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Wounded ex vivo DCs and RCs cultured on ABDSs reliably epithelialized, formed optical haze (consistent with in vivo wound healing), and maintained convex curvature. This culture technique may be adaptable to other species.

Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection.

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 µg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 µL) from serum and saliva.

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantoms and ex vivo melanoma tumour assessment.

Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.

Adherent agarose mold cultures: An in vitro platform for multi-factorial assessment of passaged chondrocyte redifferentiation.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFß1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture.

Mechanical characterisation of agarose-based resins is an important factor in ensuring robust chromatographic performance in the manufacture of biopharmaceuticals. Pressure-flow profiles are most commonly used to characterise these properties. There are a number of drawbacks with this method, including the potential need for several re-packs to achieve the desired packing quality, the impact of wall effects on experimental set up and the quantities of chromatography media and buffers required. To address these issues, we have developed a dynamic mechanical analysis (DMA) technique that characterises the mechanical properties of resins based on the viscoelasticity of a 1ml sample of slurry. This technique was conducted on seven resins with varying degrees of mechanical robustness and the results were compared to pressure-flow test results on the same resins. Results show a strong correlation between the two techniques. The most mechanically robust resin (Capto Q) had a critical velocity 3.3 times higher than the weakest (Sepharose CL-4B), whilst the DMA technique showed Capto Q to have a slurry deformation rate 8.3 times lower than Sepharose CL-4B. To ascertain whether polymer structure is indicative of mechanical strength, scanning electron microscopy images were also used to study the structural properties of each resin. Results indicate that DMA can be used as a small volume, complementary technique for the mechanical characterisation of chromatography media.

Direct detection of cysteine using functionalized BaTiO3 nanoparticles film based self-powered biosensor.

Simple, novel, and direct detection of clinically important biomolecules have continuous demand among scientific community as well as in market. Here, we report the first direct detection and facile fabrication of a cysteine-responsive, film-based, self-powered device. NH2 functionalized BaTiO3 nanoparticles (BT-NH2 NPs) suspended in a three-dimensional matrix of an agarose (Ag) film, were used for cysteine detection. BaTiO3 nanoparticles (BT NPs) semiconducting as well as piezoelectric properties were harnessed in this study. The changes in surface charge properties of the film with respect to cysteine concentrations were determined using a current-voltage (I-V) technique. The current response increased with cysteine concentration (linear concentration range=10µM-1mM). Based on the properties of the composite (BT/Ag), we created a self-powered cysteine sensor in which the output voltage from a piezoelectric nanogenerator was used to drive the sensor. The potential drop across the sensor was measured as a function of cysteine concentrations. Real-time analysis of sensor performance was carried out on urine samples by non-invasive method. This novel sensor demonstrated good selectivity, linear concentration range and detection limit of 10µM; acceptable for routine analysis.

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

A colorimetric assay for measuring iodide using Au@Ag core-shell nanoparticles coupled with Cu(2+).

Au@Ag core-shell nanoparticles (NPs) were synthesized and coupled with copper ion (Cu(2+)) for the colorimetric sensing of iodide ion (I(-)). This assay relies on the fact that the absorption spectra and the color of metallic core-shell NPs are sensitive to their chemical ingredient and dimensional core-to-shell ratio. When I(-) was added to the Au@Ag core-shell NPs-Cu(2+) system/solution, Cu(2+) can oxidize I(-) into iodine (I2), which can further oxidize silver shells to form silver iodide (AgI). The generated Au@AgI core-shell NPs led to color changes from yellow to purple, which was utilized for the colorimetric sensing of I(-). The assay only took 10 min with a lowest detectable concentration of 0.5 µM, and it exhibited excellent selectivity for I(-) over other common anions tested. Furthermore, Au@Ag core-shell NPs-Cu(2+) was embedded into agarose gels as inexpensive and portable "test strips", which were successfully used for the semi-quantitation of I(-) in dried kelps.

One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency.

A comprehensive molecular dynamics approach to protein retention modeling in ion exchange chromatography.

In downstream processing, the underlying adsorption mechanism of biomolecules to adsorbent material are still subject of extensive research. One approach to more mechanistic understanding is simulating this adsorption process and hereby the possibility to identify the parameters with strongest impact. So far this method was applied with all-atom molecular dynamics simulations of two model proteins on one cation exchanger. In this work we developed a molecular dynamics tool to simulate protein-adsorber interaction for various proteins on an anion exchanger and ran gradient elution experiments to relate the simulation results to experimental data. We were able to show that simulation results yield similar results as experimental data regarding retention behavior as well as binding orientation. We could identify arginines in case of cation exchangers and aspartic acids in case of anion exchangers as major contributors to binding.

Agarose-based microfluidic device for point-of-care concentration and detection of pathogen.

Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 µL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 µL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.

Preparation and validation of low cost microfluidic chips using a shrinking approach.

The present paper describes the production of microfluidic chips using an approach based on shrinkable biocompatible polymers (i.e. agarose) for the production of size controlled microfluidic channels. In addition, all steps of chip production were carried out using an inexpensive approach that uses low cost chemicals and equipment. The produced chips were then validated by producing monodisperse polymeric microparticles for drug delivery and hydrogel microfibers for cell embedding.

Alkanethiol-functionalized terahertz metamaterial as label-free, highly-sensitive and specific biosensor.

Specific biorecognition is essential for many biological processes, for which highly sensitive and label-free biosensors are strongly demanded. The recently developed metamaterials are a potential choice for biosensing due to their exotic properties. In the current work, a label-free and specific sensor for streptavidin-agarose (SA) was fabricated based on terahertz metamaterial functionlized by octadecanthiols and biotins. Both low and high frequency resonant modes from the metamaterials are found applicable for the detection of SA, and a redshift up to 6.76 GHz for the high frequency mode was measured in the undiluted commercial solution. The low frequency mode is attributed to inductor-capacitor (LC) oscillation, while the high frequency mode originates from the plasmonic dipole oscillator, both of which are highly sensitive to the micro-environment change. Adsorption of SA of different concentrations causes different redshifts, and the replacement of high refractive-index substrate with low refractive-index substrate can efficiently promote the sensitivity, well agreeing with the numerical simulation. Moreover, for a particular biomolecule, the sensitivity can be further improved by optimizing the metamaterial design. This method might be very helpful for desirable biorecognition in biology, medicine, and drug industry.

Fabrication and evaluation of low-cost agarose-zinc nanoporous composite matrix: influence of adsorbent density and size distribution on the performance of expanded beds.

Expanded bed adsorption (EBA), a promising and practical separation technique for adsorption of nanobioproduct/bioproduct, has been widely studied in the past two decades. The development of adsorbent with the special design for expanded bed process is a challenging course. To reduce the costs of adsorbent preparation, fine zinc powder was used as the inexpensive densifier. A series of matrices named Ag-Zn were prepared by water-in-oil emulsification method. The structure and morphology of the prepared matrix were studied by the optical microscope (OM) and scanning electron microscopy (SEM). The physical properties as a function of zinc powder ratio to agarose slurry were measured. The prepared matrices had regular spherical shape, and followed logarithmic normal size distribution with the range of 75-330 µm, mean diameter of 140.54-191.11 µm, wet density of 1.33-2.01 g/ml, water content of 0.45-0.75, porosity of 0.86-0.97 and pore size of about 40-90 nm. The bed expansion factor at the range of 2-3 was examined. The obtained results indicated that the expansion factor was decreased with increasing of matrix density. In addition, it was found that matrices with large particle size were suitable for high operation flow rate. The hydrodynamic properties were determined in expanded bed by the residence time distribution method (RTD). The effects of flow velocity, expansion factor and density of matrix on the hydrodynamic properties were also investigated. Moreover, the influence of particle size distribution on the performance of expanded bed has been studied. Therefore, three different particle size fractions (65-140, 215-280 and 65-280 µm) were assessed. The results indicated that dispersion in liquid-solid expanded beds increased with increasing flow rate and expansion factor; and matrix with a wide particle size distribution leaded to a reduced axial dispersion compared to matrices with a narrow size distribution. The axial dispersion coefficient also enhanced with the increasing of matrix density. It was found that flow rate was the most essential factor to effect on the hydrodynamic characteristics in the bed. For all the prepared matrices, the values of axial mixing coefficients (D(axl)) were smaller than 1.0 × 10⁻5 m²/s when flow velocities in expanded bed were less than 700 cm/h. All the results indicate that the prepared matrix show good expansion and stability in expanded bed; and it is suitable for expanded bed processes as an economical adsorbent.

A lithography-free procedure for fabricating three-dimensional microchannels using hydrogel molds.

We present a lithography-free procedure for fabricating intrinsically three-dimensional smooth-walled microchannels within poly(dimethylsiloxane) (PDMS) elastomer using hydrogel molds. In the fabrication process, small pieces of agarose gel ("wires" or "chips") are embedded in uncured PDMS composite, arranged in the shape of the desired microchannels, and used as molds to form the microchannels. The point of the process is that molds for creating junctions of microchannels such as T-junctions or cross-junctions can be robustly formed by simply grafting gel wires in uncured PDMS composite without using adhesive agents. The technical advantage of this method is that three-dimensional microstructures such as microchannels with circular cross sections, three-dimensionally arranged junctions or interchanges of microchannels can be flexibly designed and fabricated with a straightforward procedure without the need for any specialized equipment or layer-by-layer assemblage processes. This method provides a low-cost, green procedure for fabricating microfluidic devices and promises to make microfluidic processes more accessible and easy to implement in a variety of scientific fields.

Porous ceramic/agarose composite adsorbents for fast protein liquid chromatography.

Porous ceramic/agarose composite adsorbents were designed and prepared with silica ceramic beads and 4% agarose gel, and then functionalized with a special ligand carboxymethyl. A novel method was introduced to fabricating of the porous silica ceramic beads. The morphology of SEM shows a spherical shape and a porous structure of the ceramic beads. Nitrogen adsorption-desorption analysis gives an average pore size of 287.5 Å, a BET surface area of 29.33 m²/g and a porosity of 41.8%, respectively. Additionally, X-ray diffraction pattern indicates that the amorphous silica has been transformed into two crystal phases of quartz and cristobalite, leading to a porous and rigid skeleton and ensuring the application of the composite beads at high flow velocities. Lysozyme of hen egg-white with the activity of 12,700 U/mg was purified by the composite ion-exchanger in one step and the recovery and purification factor reaches 95.2% and 7.9, respectively.

High performance gel imaging with a commercial single lens reflex camera.

A high performance gel imaging system was constructed using a digital single lens reflex camera with epi-illumination to image 19 × 23 cm agarose gels with up to 10,000 DNA bands each. It was found to give equivalent performance to a laser scanner in this high throughput DNA fingerprinting application using the fluorophore SYBR Green(®). The specificity and sensitivity of the imager and scanner were within 1% using the same band identification software. Low and high cost color filters were also compared and it was found that with care, good results could be obtained with inexpensive dyed acrylic filters in combination with more costly dielectric interference filters, but that very poor combinations were also possible. Methods for determining resolution, dynamic range, and optical efficiency for imagers are also proposed to facilitate comparison between systems.