Chemical and Biochemical Procedures Laboratory

  1. Synthesis and investigation of multifunctional nanoparticles

Synthesis of multifunctional nanoparticles as pharmaceutics and/or biocatalysts is an important research topics.

a. Nanoparticles as drug carriers: synthesis of multifunctional drug carrier nanoparticles based on poly(amidoamin) (PAMAM) dendrimer molecules. Dendrimers are highly branched monodisperse molecules and contain tree-like branches (Fig. 1). Dendrimers have symmetric macromolecular structure with special chemical features. Pharmaceutical drug molecules, signal molecules (paint molecules, e.g. fluorescein isothiocyanate or MRI signal molecules, e.g. gadolinium-DOTA complex) and target molecules (which can recognize specific molecular patterns of damaged tissues or cells, e.g. folic acid in the case of breast cancer) are covalently bounded on the surface of the same dendrimer molecule by multi step synthesis (Fig. 2). For example, synthesis of multifunctional nanoparticles as targeted therapeutic agents 1) is started with the partial acetylation of surface amine groups of PAMAM dendrimer and 2) followed by covalent attachment of signal molecules to the free amino groups remained on the surface of the dendrimer molecule. 3) After it, target molecules, which can recognize the unhealthy cells, are conjugated covalently to the dendrimer scaffold and 4) finally drug molecules are bounded to the multifunctional complex (Fig. 2). Cancerous cells recognize this targeted drug carrier nanoparticles but healthy cells not recognize them. Therefore cancerous cells uptake the multifunctional nanoparticles with drug molecules and drug molecules kill them.

Fig 1

Fig. 1 Composition of poly(amidoamin) (PAMAM) dendrimer. Dendrimers have tree-branch-like structure (core and shells). PAMAM dendrimers can carry metal complexes, metal nanoparticles or drug molecules.

Fig 2

Fig. 2 Multifunctional carrier nanoparticle synthesized from PAMAM dendrimer as scaffold molecule. Left side: synthesis steps of multifunctional carrier. Right side: composition of multifunctional nanoparticle as drug carrier (G5: PAMAM dendrimer of 5th generation; FA: folic acid; FITC: fluorescein isothiocyanate; OH: glycidol). (Subscript numbers in parentheses mean the number of molecules bound on the dendrimer)

b. Nanoparticle as catalyzer: the biocatalyst (enzyme) is pretreated (stabilization by covering the enzyme molecule with polymer layer, see below, Fig. 4) and conjugated to nanoparticles (e.g. to magnetic nanoparticles) in order to reuse them after a cycle of biocatalytic reaction. It is investigated, which pretreatment method could increase the active time period of the biocatalyst (enzyme).

2. Pressure retarded osmosis (PRO) process

The PRO energy generation technique uses a semipermeable membrane to separate a less concentrated solution (usually known as feed solution), or solvent (for example fresh water, surface water) from a more concentrated (usually called as draw solution) and pressurized solution (for example sea water, brine water), allowing the solvent, water, to pass into the concentrated solution, through the membrane, due to the osmotic pressure gradient, across the membrane. The solvent transport increases its volume on the draw side, which can be depressurized by a hydroturbine system to produce power. A great quantity of renewable, “blue” energy can be potentially generated when waters of different salinities are mixed together. Harvesting of this energy for conversion into power can be accomplished by means of the pressure retarded osmosis (PRO). Moreover, PRO is considered a clean technology, because negligible chemical use or CO2 emissions are involved in the process of energy generation

The main point of our research activity is study of the mass transfer process (solute and solvent) taking into account all mass transfer resistances. Mass transport through a PRO (as well as FO, RO) membranes is complex one and depends on many parameters including membrane type, structure, and orientation, temperatures and compositions of the feed and draw solutions, hydraulics, etc. In osmotically driven membrane processes there are two orientations in which the membrane is utilized in these processes. The most common orientation in the PRO processes is that the active layer is in contact with the high salinity draw solution, while in FO process it is in contact with the low salinity feed solution. The solute transport in PRO process is illustrated in Fig. 3:


Fig. 3 Illustration of salt concentration profiles and giving the denotes and mass transfer coefficients in case of asymmetric membrane for pressure retarded osmosis taking into account both the external and internal polarization layers. Δπeff means the effective osmotic pressure. Its value can be determined by difference of the interfacial membrane concentrations, namely Cm-Cf. The β mass transfer coefficients (βd, βs, βf are mass transfer coefficients in presence of convection.


Fig. 4 Laboratory device for pressure retarded osmosis experiments

3. Improvement of enzyme stability by synthesis of polymer layer

New synthesis techniques are improved to create very stable enzymes with long lifetime. Enzyme molecule is covered by a very thin and porous polymer layer (Fig. 5). The synthesis route is simple: after a modification (acryloylation) on the surface of the enzyme molecule in aqueous solution (Fig.5/1.), poly(acrylamide-bisacrylamide) random spatial copolymer are synthesized around the enzyme molecule (Fig. 5/2.). These enzyme-polymer conjugates or enzyme nanoparticles could digest greater substrates e.g. cellulase enzyme nanoparticles break down cellulose to glucose, as well. These enzyme nanoparticles are stable under extremely high temperature (e.g. cellulase or beta-xylosidase enzyme nanoparticles have measurable activity for few days, at even 80 °C), where native enzymes lose their activity very quickly.

Now biodegradable and biocompatible polymer layers e.g. poly(N-isopropyl acylamide/bisacrylamide) and poly(N,N’-dimethylacrylamide/bisacrylamide) copolymers are synthesized (see Fig. 5/2.) and enzyme stability is investigated at higher temperature (80 °C).

Fig 5

Fig. 5 Synthesis of enzyme nanoparticles: each enzyme molecule is covered with a very thin, few nanometer thick polymer layer. The polymerization reaction is started from the surface of the enzyme molecule. The resulted polymer nanoparticles have enhanced lifetime (at least 20-40 times longer lifetime than that of the natural enzymes), good pH-stability and heat stability even at 80 °C.

3. Investigation of biocatalytic membrane reactors

Mostly enzyme catalytic bioreactors are investigated both theoretically and experimentally. Enzyme(s) are immobilized in the porous support layer of the asymmetric membrane and the product separation happens in the thin active layer. The biocatalytic membranes are illustrated in Fig. 6. The one-pass method is used where the substrate solution is forced through the biocatalytic membrane from the porous side, applying transmembrane pressure. Joint researches are carried out with Membrane Technology Institute in Rende (Calabria, Italy). In framework of this research collaboration hydrolysis of oleuropein into algycon and pectin into galacturonic acid were investigated.


Fig.6 Schematic graphs of the different operating modes of the biocatalytic membrane reactor; A: enzyme (E) is immobilized in the porous, support layer in an asymmetric membrane; A1, A2: entering the substrate containing solution the bicatalytic pores, depending the transmembrane pressure difference; B: enzyme is immobilized in a gel layer on the membrane surface.

Enzyme are used in its native and also in its pretreated form (discussed in the 3th section), where enzyme is covered by thin polymeric membrane, which provides more stable enzyme. Bioreactor used for microbial biochemical processes is illustrated by Fig. 7.


Fig. 7 Bioreactor for investigation of biocatalytic (microbial and enzymatic) reaction

The combination of chemical (biochemical) reaction (mostly for cases of reversible reaction) by pervaporation for removal of the product component, is also an important process, the so-called hybrid process. The laboratory scale equipment is shown in Fig. 8.

Fig 8

Fig.8 Combination of reactor by membrane separation, namely pervaporation, process

4. Synthesis of gemini surfactants (MOL Nyrt.)

The purpose of this R&D research is to develop advanced surfactant-based enhanced oil recovery (EOR) processes based upon tailoring new high-performance and cost-effective gemini surfactants.

Chemically enhanced oil recovery (EOR) and particularl surfactant injection has recently received a great deal of attention. The suggested recovery mechanisms after injecting surfactants include wettability alteration and IFT reduction. If a surfactant is properly selected according to the environmental variables-such as pressure, temperature, salinity, it can lead to more efficient enhanced recovery from an oil reservoir.

Gemini surfactants are a group of novel surfactants with more than one hydrophilic head group and hydrophobic tail group linked by a spacer at or near the head groups. Unique properties of gemini surfactants, such as low critical micelle concentration, good water solubility, unusual micelle structures and aggregation behavior, high efficiency in reducing oil/water interfacial tension, and interesting rheological properties.

The current research activities on the application of gemini surfactants in EOR.

5. Mass transfer investigation through a membrane layer

The mass transport mechanisms and their mathematical description are investigated through different types of membrane layers (asymmetric, porous, nonporous) by different applications (e.g. pervaporation, nanofiltration, reverse osmosis, pressure retarded osmosis, forward osmosis as well as mass transport accompanied by chemical-, biochemical reactions. It is studied the effect of the solution, diffusion, (constant, local coordinate and/or concentration dependent diffusivity) and convection (as effect of hydraulic pressure, osmotic pressure) on selectivity, mass transfer rate and on the membrane performance, The aim of this research works is the description of the separation efficiency of membrane process in question, the improvement of its performance, data’ supply for planning and realization of industrial processes. The concentration distribution with external mass transfer resistance is illustrated in Fig. 9.


Fig. 9 Illustration of the concentration distribution and the important variables through mass transport through a membrane layer


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