Agarose Concentration Research Articles

Concentration dependence of the sol-gel phase behavior of agarose-water system observed by the optical bubble pressure tensiometry

We have studied an expansion behavior of pressurized bubbles at the orifice of a capillary inserted in gelator-solvent (agarose-water) mixtures as a function of the gelator concentration in which the phase transition points are included. The pressure (P) -dependence of the radius of the curvature (R) of the bubbles monitored by laser beam has shown a discontinuous decrease in the exponent (m) of the experimental power law R = KΔP−m (K: constant) from 1 to 1/2 and a discontinuous increase in the average surface tension (γave) obtained from the work-area plots of the mixtures exceeding that of pure water (72.6 mN/m) at 0.02 < [agarose] < 0.03 wt%, which is attributed to the disappearance of the fluidity. The apparent surface tension (γapp = ΔP/2 R) of the system in the concentration range of 0.03–0.20 wt% has been analyzed by a modified Shuttleworth equation γapp = σ0 + τln(A/A0), where σ0 is an isotropic constant component and the second term is a surface area (A) -dependent elastic component, in which τ is the coefficient and A0 is the area of the orifice. The analysis has indicated that σ0 coincides with the γapp value of the mixture of 0.02 wt% and the second term at >0.02 wt% is the dominant component. From the appearance of the elastic component and concentration dependence of τ, the plateau of τ for the agarose-water mixtures at 0.03–0.10 wt% (Region II) has been explained to the phase separation giving two-phase mixtures of 0.02 wt% sol and 0.10 wt% gel and the upward inflection of τ at 0.10 wt% has been assigned to an increase in the elasticity of the gel with the increase of the agarose concentration in the range of >0.10 wt% (Region III). On considering the concentration dependence of the surface tension of agarose-water mixtures, the discontinuous and inflection points were assigned to the 1st- and 2nd-order phase transition concentrations of the agarose gel, respectively. Given the results with our tensiometry based on the optical bubble pressure method, distinct gelation points for other systems could be determined both mechanically and thermodynamically which will provide a diagnostic criterion of sol-gel transitions.

Restricted and Unrestricted Migration Mechanisms of Silica Nanoparticles in Agarose Gels and Their Utilization for the Separation of Binary Mixtures

The migration of SiO2 nanoparticles (NP) was investigated during agarose gel electrophoresis with various gel mesh sizes. As a model system, spherical SiO2 NP of different sizes were synthesized by Stober synthesis, and surface-modified by covalently binding different types of ligands to tailor the zeta potential as required. In the following step, agarose gels were cast with TBE (Tris/Borate/EDTA) buffer solution, thereby controlling the mesh sizes by varying agarose concentration, with higher concentrations leading to smaller mesh sizes. Additionally, the applied voltage could be varied for different electrophoretic experiments. Generally, small mesh sizes and high applied voltages lead to faster particle migration. The migration of SiO2 NP occurs under two regimes: restricted migration, where small mesh size interferes with large particles so that the gel is acting similar to a sieve; and unrestricted migration, where mesh size does not influence the particles’ migration behavior and they move accordin...

A lab-on-chip ultrasonic platform for real-time and nondestructive assessment of extracellular matrix stiffness.

Extracellular matrix (ECM) mechanical stiffness and its dynamic change is one of the main cues that directly affects the differentiation and proliferation of normal cells as well as the progression of disease processes such as fibrosis and cancer. Recent advancements in biomaterials have enabled a wide range of polymer matrices that could mimic the ECM of different tissues for a wide range of in vitro basic research and drug discovery. However, most of the technologies utilized to quantify the stiffness of such ECM are either destructive or expensive, and therefore are unsuitable for the in situ, long-term monitoring of variations in ECM stiffness for on-chip cell culture applications. This work demonstrates a novel noninvasive on-chip platform for characterization of ECM stiffness in vitro, by monitoring ultrasonic wave attenuation through the targeted material. The device is composed of a pair of millimeter scale ultrasonic transmitter and receiver transducers with the test medium placed in between them. The transmitter generates an ultrasonic wave that propagates through the material, triggers the piezoelectric receiver and generates a corresponding electrical signal. The characterization reveals a linear (r2 = 0.86) decrease in the output voltage of the piezoelectric receiver with an average sensitivity of -15.86 μV kPa-1 by increasing the stiffnesses of hydrogels (from 4.3 kPa to 308 kPa made with various dry-weight concentrations of agarose and gelatin). The ultrasonic stiffness sensing is also demonstrated to successfully monitor dynamic changes in a simulated in vitro tissue by gradually changing the polymerization density of an agarose gel, as a proof-of-concept towards future use for 3D cell culture and drug screening. In situ long-term ultrasonic signal stability and thermal assessment of the device demonstrates its high robust performance even after two days of continuous operation, with negligible (<0.5 °C) heating of the hydrogel in contact with the piezoelectric transducers. In vitro biocompatibility assessment of the device with mammary fibroblasts further assures that the materials used in the platform did not produce a toxic response and cells remained viable under the applied ultrasound signals in the device.

Mechanical characterisation of an organic phantom candidate for breast tissue.

The aim of this study is to analyse the mechanical properties of agarose, which can be used as a mechanical phantom for breast tissues. In general, tissue mimicking materials may be used to achieve a better understanding of the structure and properties of tissues and organs; this work shares these objectives. The phantom can be used as a tissue surrogate with realistic mechanical behaviour for biomechanical applications. To validate agarose as a suitable mechanical phantom for breast tissues, indentation tests were performed in homogeneous, rectangular agarose blocks. Blocks with different agarose concentrations were moulded and tested. An estimation of the material stiffness was then compared with experimental data on different breast tissues’ types found in literature. The phantom stiffness increased consistently with agarose concentration and stress. The results show that agarose-based mechanical phantoms of stiffer tissues require higher agarose concentrations (0.5% and 0.6%). In contrast, normal tissues can be mimicked with 0.3% and 0.4% of agarose. In addition, it was observed that preconditioning affects the mechanical properties of the gel, as it had already been shown in literature for breast tissues.

Multilayered microcasting of agarose–collagen composites for neurovascular modeling

The in vitro fabrication of vascular networks is one of the most complex challenges currently faced in tissue engineering. We describe a method to create multi-layered, cell-laden hydrogel microstructures with coaxial geometries and heterogeneous elastic moduli. The technique can be used to build in vitro vascular structures that are fully embedded in physiologically realistic hydrogels. Our technique eliminates rigid polymeric surfaces from the vicinity of the cells—overcoming a limitation of many microfluidic models—and allows layers of multiple cell types to be defined with tailored ECM composition and stiffness, and in direct contact with each other. We demonstrate channels with internal diameters as small as 175 ​μm, and agarose–collagen (AC) gels whose Young’s moduli range from 1.4–8.3 ​kPa. We also show co-axial geometries with layer thicknesses as small as 125 ​μm. One potential application of such structures is to simulate brain microvasculature. Towards this goal, the composition and mechanical properties of the composite AC hydrogels are optimized for cell viability and biological performance in both 2D and 3D culture. Seven-day viability of human microvascular endothelial cells (HMECs) and SY5Y glial cells is found to be maximized with a collagen content of 0.05% (w/v) when agarose content ranges between 0.25% and 1% (w/v). Additionally, we quantify the roles of type I bovine and rat-tail collagen, Matrigel, and poly-d-lysine–collagen–Matrigel coatings in promoting HMEC spreading, proliferation and confluence. 3D triple-layer vascular constructs have been fabricated, composed of a cannular monolayer of HMECs surrounded by two regions of SY5Ys with differing spatial densities. The endothelia are confluent and maintain trans-endothelial electrical resistance (TEER) values around 300 ​Ω ​cm2 over 11.5 days. This prototype opens the way for intricate multi-luminal blood vessels to be fabricated in vitro.

Crystallization of Sulfathiazole in Gel: Polymorph Selectivity and Cross-Nucleation

Form II, Form III, and Form IV of sulfathiazole (STZ) crystallized simultaneously from water by a cooling crystallization method. Thus, how to obtain the pure form of STZ represents a major challenge during the sulfathiazole crystallization process. Here we report that the pure Form III or Form IV of STZ can be selectively grown in agarose matrix by only adjusting the concentration of agarose. Interestingly, at a specific concentration range of agarose, the cross-nucleation of Form III on Form IV was observed. Furthermore, the mechanisms of agarose that affect the process of cooling crystallization of STZ were investigated, which indicated that the crystalline outcomes correlate strongly with the state of agarose gel that existed in solution. The highly reproducible and controllable results will help to broaden the application of gel in controlling crystallization of polymorph in the pharmaceutical industry. It also can be used as a guide for polymorphic screening of STZ from cooling crystallization.

Boron nitride/agarose hydrogel composites with high thermal conductivities

Hydrogels are cross-linked polymers suitable for various applications, but the thermal conductivities of hydrogel-based composites have not been thoroughly investigated. In this study, agarose hydrogel-based composites with various boron nitride (BN) fillers were synthesized and their thermal conductivities were systematically investigated. With the increase in the agarose content from 1.5 wt% to 3.0 wt%, the thermal conductivity of the composite decreased. The composites with BN micropowder had larger thermal conductivities than those of the composites with BN nanopowder at the same filler loading, as the BN micropowder provided better thermal conduction pathways in the hydrogel matrix than those provided by the nanopowder. The maximum thermal conductivity of 2.69 W·m−1·K−1 was achieved when 15 wt% microscale BN fillers were added into 1.5 wt% agarose hydrogel, which was 3.5 times larger than that of the pure agarose hydrogel. Additionally, a theoretical model was used to calculate the thermal conductivities of the BN/agarose hydrogel composites; a good agreement was achieved between the experimental and fitting ones. This study demonstrated that the thermal conductivities of hydrogel-based materials can be efficiently and significantly enhanced using BN fillers.

Microstructural evolution of mullite nanofibrous aerogels with different ice crystal growth inhibitors

Mullite nanofibrous aerogels with low density, low thermal conductivity and high service temperature (as high as 1400 °C) was fabricated by the freeze-casting method, and two different kinds of ice crystal growth inhibitors, glycerol as the cryoprotector and agarose as the gel-former, were adopted respectively to influence the growth of ice crystal, thereby adjusting the sample microstructure (including the pore size and the pore distribution). The addition of glycerol could influence the growth of the ice crystal, leading to the pore structure changing from the cellular pores to the spherical pores and the decrease of the large pore size, consequently resulting in the increase of the sample density and compressive strength. The addition of agarose could inhibit the growth of the ice crystal due to the formation of gel network, resulting in the disappearance of the large pores (cellular pores). The sample with high agarose content exhibited a relatively high compressive strength of 252.41 kPa due to high density and uniform pore structure. The research of this work provides an effective method to regulate the properties of nanofibrous aerogel to meet the demand of different applications.

In vitro assessment of stiffness-dependent histotripsy bubble cloud activity in gel phantoms and blood clots

As a bubble-based ablative therapy, the efficacy of histotripsy has been demonstrated in healthy or acutely diseased models. Chronic conditions associated with stiff tissues may require additional bubble activity prior to histotripsy liquefaction. In this study, histotripsy pulses were generated in agarose phantoms of Young’s moduli ranging from 12.3 to 142 kPa, and in vitro clot models with mild and strong platelet-activated retraction. Bubble cloud emissions were tracked with passive cavitation imaging, and the threshold acoustic power associated with phantom liquefaction was extracted with receiver operator characteristic analysis. The power of histotripsy-generated emissions and the degree of liquefaction were tabulated for both clot models. For the agarose phantoms, the acoustic power associated with liquefaction increased with Young’s modulus. When grouped based on agarose concentration, only two arms displayed a significant difference in the liquefaction threshold acoustic power (22.1 kPa versus 142 kPa Young’s modulus). The bubble cloud dynamics tracked with passive cavitation imaging indicated no strong changes in the bubble dynamics based on the phantom stiffness. For identical histotripsy exposure, the power of acoustic emissions and degree of clot lysis did not vary based on the clot model. Overall, these results indicate that a fixed threshold acoustic power mapped with passive cavitation imaging can be utilized for predicting histotripsy liquefaction over a wide range of tissue stiffness.

Improved Electrical Performance By Separating Enzymes By Agarose in Bi-Enzyme Electrode for Biofuel Cells

Equipped with tiny sensors, insects can scout out buildings filled with noxious chemicals, check under rubble after an earthquake and go places no human spy over could. Power source is required to power the electronics attached to insects. Recently, insect-mountable enzyme fuel cells (EFCs) have been presented, which utilize trehalose, the main sugar of insect hemolymph. The anode system composes of two enzymes, glucose oxidase (GOD) and trehalase (THL), and enzyme mixture was used for fabrication of anode in reported EFCs. In this study, however, we demonstrated that THL prevented electron transfer from enzyme layer to electrodes and separating it from GOD can improve the performance of bi-enzyme electrode. Here, two enzymes were immobilized on gold electrode by physical adsorption and they were separated by agarose gel. Osmium-based polymers were used as electron transfer mediators. The optimal concentration of agarose was determined. The effects of GOD-Os layer and THL loading were examined. The performances were compared for electrodes with or without single walled carbon nanotubes (SWCNTs) in the GOD layers. The electrical properties were measured using electrochemical impedance spectroscopy and cyclic voltammetry. The cell performance was evaluated with optimized anode and cathode covered with bilirubin oxidase. The maximum power density was 1.23 ± 0.04 mW/cm2.

Correlation of quantitative conductivity mapping and total tissue sodium concentration at 3T/4T.

To investigate the correlation between electrical conductivity and sodium concentration, both measured in vivo, in the human brain. Conductivity measurements were performed on samples with different sodium (Na+ ) and agarose concentrations using a dielectric probe, and the correlation between conductivity and Na+ content was evaluated. Subsequently, brain conductivity and total Na+ content maps were measured in 8 healthy subjects using phase-based MREPT and sodium MRI, respectively. After co-registration and spatial normalization to the 1 mm 152 MNI brain atlas, the relationship between conductivity and tissue sodium concentration (TSC) was examined within different brain regions. The conductivities of agarose gels increased linearly with NaCl concentration, while remaining almost independent of agarose content. When measured in healthy subjects, conductivities showed positive correlation with total tissue sodium concentration (R = 0.39, P < 0.005). The same trend was found in gray matter (R = 0.36, P < 0.005) and in white matter (R = 0.28, P < 0.05). Tissue conductivity shows a positive correlation with total sodium concentration. Conductivity might serve as a novel technique to visualize the total tissue electrolyte concentration, although refinements in the consideration of e.g., tissue water content, would be necessary to improve the quantitative value.

Antimicrobial hydrogels with controllable mechanical properties for biomedical application

Abstract

Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies

The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage – strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored – and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.

A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis

CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4–6% agarose gel electrophoresis. WT/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies.

A continuum of components: Flexible fast fraction mapping in sodium MRI.

Parameter mapping in sodium MRI data is challenging due to inherently low SNR and spatial resolution, prompting the need to employ robust models and estimation techniques. This work aims to develop a continuum model of sodium -decay to overcome the limitations of the commonly employed bi-exponential models. Estimates of mean -decay and fast component fraction in tissue are emergent from the inferred continuum model. A closed-form continuum model was derived assuming a gamma distribution of components. Sodium MRI was performed on four healthy human subjects and a phantom consisting of closely packed vials filled with an aqueous solution of varying sodium and agarose concentrations. The continuum model was applied to the phantom and in vivo human multi-echo 7T data. Parameter maps by voxelwise model-fitting were obtained. The continuum model demonstrated comparable estimation performance to the bi-exponential model. The parameter maps provided improved contrast between tissue structures. The fast component fraction, an indicator of the heterogeneity of localised sodium motion regimes in tissue, was zero in CSF and high in WM structures. The continuum distribution model provides high quality, high contrast parameter maps, and informative voxelwise estimates of the relative weighting between fast and slow decay components.

The effect of bioresorbable additives and micro-bioobjects on gel formation, stabilization and thermophysical properties

The same properties of agarose gels containing neutral bioresorbable additives and living microorganisms which are important for use in additive technologies of bioreactors creation were considered. Data on the kinetics of gel formation from the solution during cooling were obtained by spectroscopic measurement by measuring the shift of the maximum spectrum of light passing through the gel, depending on the temperature. The dynamics of aging was investigated for gels of different concentrations of agarose, bioresorbable additives and living cells. The time dependences of the decrease in the optical transparency of such gels during the aging process, characterizing the changes in their structure, were obtained. Special attention was paid to the effect of liquid evaporation from gels in the process of gel formation and during long-term storage on relaxation processes leading to their spontaneous increase in density. Experiments were performed to determine the dynamics of the temperature fields simultaneously with heat flux measurements during the formation of studied gels with different concentrations. On the basis of the obtained experimental data and previously developed method, the thermophysical coefficients of agarose gels containing an admixture of starch and living yeast cells were calculated.

Study of the viscoelastic properties of the agarose gel

Abstract Agarose is a biopolymer extracted from the red marine alga (Rhodophycees), it is widely used in agribusiness, pharmaceutical industry or as electrophoresis. The solution of this biopolymer in water gives rise to a more or less rigid solid mass called gel. This gel can be described as a matrix generating a macromolecular three-dimensional network. In this work, we exploit the experimental measurements of the stress undergone by the gel and the underlying deformation as a function of the different concentrations of the agarose to determine the creep function of this polymer in a linear regime according to the Boltzmann superposition principle. It turns out from the calculations that the gel is subjected to a delayed elasticity governed by Voigt’s analog model. This model allowed us to find the elastic modulus of the gel, and to determine the delay time and viscosity as function s of agarose concentrations.

Cryoprotectant-Free Freezing of Cells Using Liquid Marbles Filled with Hydrogel.

Cryopreservation without cryoprotectant remains a significant challenge for the re-establishment of cell culture after freeze-thaw. Thus, finding an alternative and a simple cryopreservation method is necessary. Liquid marble (LM)-based digital microfluidics is a promising approach for cryoprotectant-free cryopreservation. However, the use of this platform to efficiently preserve samples with low cell density and well-controlled serum concentrations has not been investigated. We addressed this issue by embedding an agarose-containing fetal bovine serum (FBS) inside the LM. A low density of 500 cells/μL of murine 3T3 cells was selected for evaluating the postcryogenic survivability. The effects on the post-thaw cell viability of the concentration of agarose, the amount of FBS inside the agarose, and the volume of the LM were investigated systematically. This paper also presents an analysis on the changes in shape and crack size of post-thawed agarose. The results revealed that the embedded agarose gel serves as a controlled release mechanism of FBS and significantly improves cell viability. Post-thaw recovery sustains major cellular features, such as viability, cell adhesion, and morphology. The platform technology reported here opens up new possibilities to cryopreserve rare biological samples without the toxicity risk of cryoprotectants.

In-gel study of the effect of magnetic nanoparticles immobilization on their heating efficiency for application in Magnetic Fluid Hyperthermia

Abstract Recent studies on magnetic nanoparticles (MNPs) used for Magnetic Fluid Hyperthermia treatments have shown that Brownian rotation is suppressed when they are confined within a cell. To investigate this effect we conducted a systematic study of the Specific Absorption Rate (SAR) of colloidal suspensions of MNPs in water and gels at different agarose concentration. SAR measurements were conducted by varying the frequency (f = 110–990 kHz) and amplitude (up to 17 kA/m) of the applied alternating magnetic field (AMF). MNP samples with different diameter (d = 10, 14, and 18 nm) were used. Our results show that Neel relaxation dominates SAR with negligible contribution from Brownian motion for smaller MNPs (d = 10 nm). For the largest MNPs (d = 18 nm) we observed a more significant SAR decrease in gel suspensions as compared to those in solution. In particular, when applying AMFs as the ones used in a clinical setting (16.2 kA/m at f = 110 kHz), we measured SAR value of 67 W/g in solution and 25 W/g in gel. This experimental finding demonstrates that investigation of MNPs properties should be conducted in media with viscosity similar to the one found in mammalian tissues.

Towards optimized MRI contrast agents for implant engineering: Clustering and immobilization effects of magnetic nanoparticles

Abstract Magnetic nanoparticles (MNP) have been successfully used as additives for the fabrication of implants such as hernia implants or vascular grafts in order to enable their in-vivo visualization with magnetic resonance imaging (MRI). However, functionality and long term stability of such implants were not quantitatively assessed until now. Reliable assessment of functionality is related not only to the determination of MNP concentration, but also to the impact of aggregation and immobilization of MNP inside the implant material on the MRI signal. In this regard, novel models must be developed which describe the relation between proton relaxation and both MNP clustering and mobility of MNP inside the implants. In this study, we experimentally quantify the transverse relaxation dependence on MNP size and MNP clusters, confirming theoretical descriptions. We identify three MNP size ranges for which different proton relaxation trends occur: One for which relaxivity increases with size (up to approx. 75 nm), a second for which relaxivity is constant (from 75 nm to 130 nm) and a third for which relaxivity decreases (from 130 nm to 220 nm). Further, we describe the impact of gradual MNP immobilization in agarose gels on relaxivity for three MNP types representing either of the identified size ranges. For all MNP types, we observe an increase of relaxivity with agarose content up to an MNP type specific maximum value. The relative rise of relaxivity is higher for MNP with larger sizes. The highest increase of the transverse relaxivity from 240 mM-1s−1 to 1000 mM-1 s−1 is achieved for MNP clusters after immobilization in a gel with 7%(w/w) agarose. The effects of MNP clustering and immobilization on relaxivity are valuable information for the engineering of implants with different contrast properties in MRI. Further, the relation between MNP immobilization and relaxivity values may serve as a basis for future non-invasive assessment of changes in implant functionality by MRI measurements.