Making greener cellulose hydrogel
The use of Microalgae as a renewable resource has attracted large interest due to their potential for use in the renewable energy, biopharmaceutical, and nutraceutical industries. Microalgae are renewable, sustainable, and economical sources of biofuels, bioactive medicinal products, and food ingredients. Recently, researchers around Professor Avtar Singh Matharu from the Green Chemistry Centre of Excellence in York, United Kingdom have developed a green process to produce cellulose hydrogel using native and spent microalgae. Cellulose extraction from microalgae without any pre-treatment is difficult, since the microalgal cell wall is very thick and rigid, but the researchers have developed a protocol for Microwave-assisted extraction (MAE) which efficiently disrupts algal cell walls aiding the production of defibrillated celluloses that are then further processed to form cellulose-based hydrogels. Cellulose hydrogel is an emerging biomaterial with many different applications such as use as cosmetics, lubricants and within medical biotechnology.
In a recent publication, the researchers at the Green Chemistry Centre of Excellence with Frederick L. Zitzman as lead-author used Carbotrace 480 to benchmark the MAE process and investigate why only MAE at 220°C yields high-quality cellulose hydrogels. Zitzmann et al. performed MAE at different temperatures with native and spent microalgae and used Carbotrace 480 to visualize cellulose in all samples. Carbotrace 480 has its emission maximum in the region of 480 nm. Upon binding to cellulose, the emission peak shifts approx. 20 nm and the intensity in emission increases by around a factor of two. Its spectral properties make Carbotrace 480 the ideal probe for investigating cellulose in microalgae since it allows for spectral unmixing with the autofluorescence of the biomass peaking at 670 nm. Carbotrace 480 shown to be useful to determine differences in morphology between native and spent biomass showing the native biomass as an array of microalgae cells, each with a ring of cellulose encapsulating the cells and the spent biomass as an array of smudged and smashed cells with irregular shapes and a more even distribution of cellulose. Carbotrace 480 also showed clearly an increase in cellulose fluorescence when MAE was performed at higher temperatures in native and spent biomass (see figure). The Carbotrace 480 signal correlated very well with the observed capability for hydrogel formation. Therefore, Carbotrace has proven to be exceptionally suited to aid the exploitation of potential renewable resources and the development of greener processes for hydrogel production.
Image: Carbotrace 480 labeling cellulose in biomass (green) with biomass autofluorescence (red). From Zitzmann et al. (CC BY 4.0)).
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Carbohydrate content and elasticity of cell walls in elongating maize root
Investigation of plant growth and development is now more than ever an important field of study following the many challenges faced in agriculture. Accurate methods and computational models of plant cell- and organ change during growth are therefore essential to tackle problems like increased food demand in the wake of climate change. Researchers at Kazan Institute of Biochemistry and Biophysics aimed to determine if the mechanical properties of root tissues can control growth. Since growth rate and direction is determined by mechanical properties of plant cell walls, they used an approach based on atomic force microscopy (AFM) to study cell wall elasticity and turgor pressure in elongating primary root tissues of maize. They determined the elasticity of maize root cell walls in the four root development zones called meristem, early elongation-, active elongation-, and late elongation zone. Interestingly, they found that cell walls in root tissues show different patterns of elasticity along the length of the root. Some cell walls have increased stiffness only in the late elongation zone (AAAB type), others had increased stiffness in meristem and late elongation zones (BAAB type) and again others showed the same pattern, but the stiffness was even more increased in the late elongation zone than in the meristem (BAAC type). To understand if there is a link between the cell wall’s elasticity and its polysaccharide composition, the researchers coupled AFM analysis with fluorescent labelling of cell wall polysaccharides. Carbotrace 680 was used for detection and quantification of cellulose.
Image: Carbotrace 680 labelling cellulose in maize root. Image courtesy of Anna Petrova. Click here to read more about the author's experience with Carbotrace 680) along with fluorescent labelled antibodies.
The signal intensity of Carbotrace 680 and the polysaccharide-binding antibodies was plotted against the elasticity in the different developmental zones along the length of the root. In contradiction to previous studies, no correlation was observed. The presence of the polysaccharides studied could therefore not be used to predict the elastic moduli of cell walls. However, by normalizing the fluorescence intensity of the fluorescent antibodies binding to hemicelluloses to that of Carbotrace 680 binding to cellulose, they revealed that a reduction in the degree of homogalacturonan methylation was coupled with a transition from division to elongation and the overall decrease in the elastic modulus of cell walls between the meristem and early elongation zone of maize root. Further, the authors were able to show that cellulose and galactoxyloglucan contents in cell walls might be important for growth patterns in developing roots. Understanding how environmental factors can influence root growth and, eventually, the ability to predict and engineer certain growth properties might be an essential step to face current and future challenges. There is a high demand for increased biomass to feed the world's population considering the effects of global warming and following a global initiative to replace fossil fuels with renewable resources. At Ebba Biotech, we are extremely proud that Carbotrace contributes to research looking into carbohydrate content of plant cell walls on a subcellular level.
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Breaking barriers in early detection of cancer
The ability to detect and isolate circulating tumor cells from blood could be a life-saving diagnostic tool for early detection of cancer and downstream analysis of tumor growth and metastasis. Researchers from the lab of Aman Russom at the Royal Institute of Technology (KTH) in Sweden have engineered a microfluidic device which captures circulating tumor cells from whole blood using antibodies immobilized on a cellulose nanofibril substrate. Using the device, circulating tumor cells were enriched and captured from whole blood. Importantly, 94.5 % of the captured tumor cells could be released from the device and isolated for downstream analysis. The innovation making such a high release efficiency possible lies in a layer-by-layer assembly of cellulose nanofibrils which serves as substrate for the immobilized antibodies. Using Carbotrace 680, the layer-by-layer assembly was carefully tuned to prevent unspecific binding of antibodies and to optimize the release of captured tumor cells during enzymatic degradation of the cellulose substrate. With the help of Carbotrace 680, the researchers were able to visualize the amount of cellulose in the microfluidic channel during the coating procedure and during enzymatic degradation of the cellulose nanofibrils. That means they were in control of the experimental conditions for optimal antibody immobilization and cell release at every step of development. Thus, they were able to determine the optimal number of layers for capturing and releasing cancer cells. The study, published in the journal Nanoscale, represents a real break-through in cancer diagnostics using a microfluidic device. Cellulose nanofibrils are featured as outstanding, bio- degradable material for capture and release of cells in diagnostic microelectromechanical systems (MEMS). Using Carbotrace to visualize the amount of cellulose nanofibril assemblies therefore adds a new level of precision to the engineering and optimization of these devices.
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Carbotrace 540 was used to determine purity of cellulose extracted from sea lettuce
Researchers from the Royal Institute of Technology and Karolinska Institutet in Sweden reported about an environmentally friendly process to obtain pure cellulose nanofibrils from the green macroalgae ulva lactuca. Cellulose was extracted by sequential treatment with ethanol, hydrogen peroxide, sodium hydroxide, and hydrochloric acid. A mechanical homogenization process was used to disintegrate the cellulose-rich fraction into cellulose nanofibrils. Analysis of the monosugars reveal that the extracted cellulose contains mainly glucose, but also a fraction of xylose. Carbotrace 540 allowed the researchers to analyze the carbohydrates in its polymeric state and was key to uncover that the macroalgae contain a mixture between highly pure and a xylose-glucose polysaccharide, such as xyloglucan.
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Carbotrace-like molecules allow highly detailed visualization of cellulose in plant cells
A study from 2018 published in Nature Scientific Reports used a Carbotrace-like molecule to visualize the location and structure of cellulose in plant cells. Applying the molecule to thin slices of onion in a simple procedure resulted in highly fluorescent cell walls and allowed to obtain detailed 3D visualization of perforated sieve plates and plasmodesmata in onion cells. The fluorescence of chlorophyll containing chloroblasts and lignin structures were easily distinguished from the fluorescent Carbotrace-like molecule. The researchers found that the molecule bound with a high specificity to β-linked glucans (polysaccharides derived from glucose) with a degree of polymerization > 8. Thus β-linked glucans like cellulose and laminarin were clearly distinguished from α-linked glucans like amylose, amylopectin, glycogen or dextran. For the first time, non-disruptive carbohydrate analysis of glucans can be performed using a specific fluorescent label for β-linked glucans. Many β-linked glucans are medically important and represent drug-targets for antifungal medications. Moreover, extraction techniques for renewable resources rely heavily on the chemical composition of their raw materials. Therefore, Carbotrace and Carbotrace-like molecules represent a quantum leap for bio-refinery of renewable resources and bio-fuels from plant-derived materials.
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Carbotrace 680 for carbohydrate anatomical mapping
In a research paper published in Cellulose, our structure-responsive optotracer molecule Carbotrace 680 was used to demonstrate the potential of optotracing for carbohydrate anatomical mapping and spectral imaging. As an example - to show the utility and ease of the new technology - Carbotrace 680 was applied to thinly sliced potato samples and then imaged with a fluorescence microscope. Strikingly, Carbotrace 680 bound to cellulose in the cell wall exhibited a unique fluorescent spectrum which was easily separated from the fluorescent spectrum of the molecule when bound to amylose and amylopectin in the potato's starch granules.
Image: The acquired image elegantly shows cell walls in great detail labeled yellow and starch granules labeled green. Evidently, the image made an impression with the editors of the "Cellulose" journal, since it was selected to appear on the cover of the 26(7) edition.
Testing a variety of glucans (polysaccharides made up of glucose) with different types of glycosidic linkages by acquiring the optical spectrum of Carbotrace 680 when bound to the carbohydrate, it was found that Carbotrace 680 differentiates α(1-3), α(1-4), α(1-6), β(1-3), β(1-4) and β(1-6) linked glucans at the molecular level and allows differentiation of cellulose and laminarin from amylose, amylopectin, glycogen and dextran. As starch is an important carrier material for drug formulations, the structure-responsive optotracer Carbotrace 680 was further applied for analyzing heat-induced swelling of starch granules and was proposed as a tool for quality control of starch-containing materials by monitoring its structural changes during production and packaging. The study describes the use of optotracing for anatomical mapping of plant tissue as well as spectral imaging and shows the potential for Carbotrace as an important tool in packaging and production of foods and drugs and in biorefinery applications for renewable materials and biofuels.